text:
Copyright © 2006 Elsevier Inc. All rights reserved.
Molecular Cell, Volume 21, Issue 2, 201-214, 20 January 2006
Add/View Comments (0)H2AX Prevents DNA Breaks from Progressing to Chromosome Breaks and Translocations
Sonia Franco1, Monica Gostissa1, Shan Zha1, David B. Lombard1, 2, Michael M. Murphy1, Ali A. Zarrin1, Catherine Yan1, Suprawee Tepsuporn1, Julio C. Morales3, Melissa M. Adams3, Zhenkun Lou4, Craig H. Bassing5, John P. Manis6, Junjie Chen4, Phillip B. Carpenter3 and Frederick W. Alt1, ,
1 Howard Hughes Medical Institute, The Children's Hospital, Department of Genetics, Harvard Medical School and the CBR Institute for Biomedical Research, Boston, Massachusetts 02115
2 Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115
3 Department of Biochemistry and Molecular Biology, University of Texas Health Sciences Center, Houston, Texas 77225
4 Department of Oncology, Mayo Clinic, Rochester, Minnesota 55905
5 Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Department of Cancer Biology and the Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
6 Joint Program in Transfusion Medicine and Department of Laboratory Medicine, Children's Hospital, Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115
- Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations.
H2AX is a mammalian histone H2A variant that undergoes rapid phosphorylation of its carboxy-terminal SQE motif by ATM and other PIKK kinases to form γ-H2AX over large chromatin domains surrounding DSBs (Rogakou et al., 1999). H2AX-deficient mammalian cells have defective chromosomal DSB repair, marked ionizing radiation sensitivity, and increased genomic instability (Bassing et al., 2002,Celeste et al., 2002), yet H2AX-deficient mice have little cancer predisposition. However, combined deficiency for H2AX and the p53 tumor suppressor, which monitors DSBs via cell cycle checkpoints and signals either arrest or apoptosis (Vogelstein et al., 2000), leads to dramatically increased cancer incidence, including T and B lineage lymphomas (Bassing et al., 2003,Celeste et al., 2003a). With respect to DSB repair defects, H2AX-deficient mammalian cells have impaired homologous recombination (Celeste et al., 2002,Xie et al., 2004). However, while H2A phosphorylation in yeast has been shown to facilitate DSB repair via nonhomologous DNA end joining (NHEJ) (Downs et al., 2000), there has been no direct evidence for an H2AX role in end joining in mammalian cells.
In developing lymphocytes, the RAG endonuclease generates DSBs at variable (V), diversity (D), and joining (J) gene segments, which are then joined by NHEJ to effect V(D)J recombination (Dudley et al., 2005). While H2AX deficiency does not measurably impair V(D)J recombination in plasmid substrates (Bassing et al., 2002,Celeste et al., 2002), a role at the chromosomal level is indicated by the occurrence of RAG-dependent γ-H2AX foci at T cell receptor loci in developing T lymphocytes (Chen et al., 2000) and findings that combined deficiency for H2AX and p53 promotes B lymphomas with translocations involving the immunoglobulin heavy chain (IgH) JH locus (Bassing et al., 2003,Celeste et al., 2003a). The murine IgH locus (Igh) harbors a series of eight Igh constant region (CH) exons (CH genes), embedded in an approximately 200 kb region downstream of the V, D, and J segments. Mature B cells can exchange the CH gene first expressed in development (Cμ) for a different, downstream CH gene (e.g., Cγ, Cɛ, Cα) via the mature B cell-specific Igh class switch recombination (CSR) process. CSR requires activation-induced deaminase (AID) (Muramatsu et al., 2000), which is thought to lead to generation of DSBs in large, repetitive switch (S) regions that lie upstream of individual CH genes, with joining of two different S regions via end joining leading to replacement of Cμ with a downstream CH gene to complete CSR (Honjo et al., 2004). Notably, γ-H2AX foci appear at the IgH locus in an AID-dependent fashion (Petersen et al., 2001), and H2AX deficiency substantially impairs chromosomal CSR (Celeste et al., 2002). Defective CSR in H2AX-deficient B cells has been suggested to reflect a role for H2AX in promoting long-range synapses of different S regions (Reina-San-Martin et al., 2003). However, some B-lineage lymphomas in mice with mutations in H2AX and p53 harbor clonal chromosomal translocations with breakpoints in or near S regions, suggesting a potential role for H2AX in suppressing inappropriate end joining during CSR (Bassing and Alt, 2004,Bassing et al., 2003).
In response to DSBs, ATM also phosphorylates other checkpoint/repair proteins including MDC1, 53BP1, and NBS1, which specifically bind the phosphorylated SQE motif of H2AX (Kobayashi et al., 2002,Stewart et al., 2003,Stucki et al., 2005,Ward et al., 2003a). This process results in a rapid, hierarchical assembly of these and other factors over γ-H2AX and the formation of multiprotein complexes (foci) around DSBs. Although generation of γ-H2AX is not essential for initial recruitment of such factors to DSBs (Celeste et al., 2003b), it is required for formation of normal, extended foci (Bassing et al., 2002,Celeste et al., 2002) and may function by providing a docking site for DSB response factors and/or by altering chromatin structure to allow their accumulation (Celeste et al., 2003b,Fernandez-Capetillo et al., 2003). Recent studies have elucidated a complex interplay between DSB response factors around DNA breaks. For example, 53BP1 may sense changes in chromatin structure via ability to interact with constitutively methylated lysine 79 (K79) of histone H3, allowing it to operate in the DSB response both upstream and downstream of ATM activation (Huyen et al., 2004). However, accumulation of 53BP1 within DSB-induced foci depends both on γ-H2AX and MDC1 (Bekker-Jensen et al., 2005,Celeste et al., 2003b,Fernandez-Capetillo et al., 2002,Stewart et al., 2003). In this regard, MDC1 appears to interact with γ-H2AX and ATM to create a positive feedback loop that propagates H2AX phosphorylation from the DSB site and amplifies the damage signal (Lou et al., 2006 [this issue of Molecular Cell]).
The localization of DSB response factors to DSBs and their accumulation in γ-H2AX foci has been suggested to function in cell cycle checkpoint responses, recruitment/activation of repair proteins, and/or tethering broken DNA ends prior to repair (reviewed in [Bassing and Alt, 2004]). In this context, mice deficient for ATM, 53BP1, NBS1, MDC1, and H2AX share a number of phenotypes potentially related to the defective DSB response, including genomic instability, DNA repair defects, and radiation sensitivity. Moreover, defects in MDC1, ATM, 53BP1, and NBS1, like H2AX deficiency, all lead to impaired CSR, albeit to varying degrees (Kracker et al., 2005,Lumsden et al., 2004,Manis et al., 2004,Reina-San-Martin et al., 2004,Reina-San-Martin et al., 2005,Ward et al., 2004,Lou et al., 2006). The requirement for all of these DSB response factors for normal CSR raises the possibility that they may have overlapping functions in the synapses or joining phase of this process (Reina-San-Martin et al., 2003,Manis et al., 2004,Bassing and Alt, 2004,Fernandez-Capetillo et al., 2004), as outlined above for H2AX.
Experimental systems in which molecular events on both sides of a defined chromosomal DSB can be characterized in a temporal and spatial manner have yielded novel insights into the function of the yeast H2AX homolog (H2A), including the role of cohesin recruitment via phosphorylated H2A in DSB repair via homologous recombination (reviewed in [van Attikum and Gasser, 2005]). However, as many repair factors that functionally interact with γ-H2AX in mammalian cells lack clear yeast homologs, it is unclear how far such studies can be generalized. As a high percentage of activated B cells undergo CSR over a few cell divisions, we theorized that this system could provide a useful experimental means of tracking the fate of DNA ends on each side of a region-specific DSB within a population of mammalian cells. Herein, we employ this approach to demonstrate that, in a process independent of p53, H2AX suppresses the progression of AID-dependent DSBs at the IgH locus to chromosomal breaks and translocations. Moreover, we find that ATM, 53BP1, and MDC1 have similar functions in suppressing chromosomal breaks and translocations during CSR.
Frequent Chromosome and Chromatid Breaks in H2AX−/− B Cells Activated for CSR
Treatment of cultured splenic IgM+ B cells with specific activators (such as LPS or α-CD40) plus cytokines (such as IL-4) activates the cells to divide, upregulate AID expression, and, after several cell divisions over 3 or 4 days, induce DSBs in targeted S regions and undergo CSR (Rush et al., 2005). Under such conditions, 20%–30% or more of wild-type (wt) B cells may undergo CSR, as evidenced by surface expression of downstream Igh isotypes such as IgG1 (see Figure S1 in the Supplemental Data available with this article online). The mouse IgH locus spans several megabases in the telomeric portion of chromosome 12 with the variable region gene (VH) segments lying closest to the telomere and the CH genes, which undergo CSR, lying more centromeric (Figure 1A, left) (Riblet, 2004). Thus, fluorescence in situ hybridization (FISH) analyses with probes that recognize the telomeres themselves or sequences just upstream of the VH domain, along with a probe that recognizes sequences immediately downstream of the CH genes, allows testing for chromosomal breaks or translocations specifically within Igh in B cells activated for CSR (Figures 1A and 2A).
We first employed spectral karyotyping (SKY) and a FISH assay that combines DAPI staining with a telomere-specific peptide nucleic acid (PNA) probe (T-FISH) to assay metaphase chromosomes for instability in wt and H2AX−/− B cells activated for CSR. Normal mouse metaphase chromosomes have four telomeres, with one at each end of the two chromatids, capping their long (q) and very short (p) arms (Figure 1A). Because the shortest mouse telomere is about 10 kbp and our assay is sensitive to telomere signals below 1 kbp, we usually detect the expected four telomere signals in every chromosome of a normal mouse metaphase. Loss of a telomere signal on both chromatids of a given arm (a chromosome break) is consistent with replication of a lesion that occurs in a prereplicative cell cycle phase, whereas loss of a telomere on a single chromatid of a given arm (a chromatid break) most commonly represents a postreplicative lesion.
Consistent with earlier findings on an independent line of H2AX-deficient mice (Celeste et al., 2002), we find that CSR in H2AX−/− B cells is reduced to levels that are 20%–50% those of wt (Figure S1 ). Strikingly, however, we further observed dramatically increased levels of chromosomal aberrations in LPS- or α-CD40/IL-4-stimulated H2AX−/− B cells, as compared to H2AX+/− or wt controls (Table 1, Figure S2 ). These general chromosomal aberrations were nearly equally divided into chromosome and chromatid breaks (Table 1, Figure S2 K), indicating that they represent defective repair of DSBs introduced throughout the cell cycle. Overall, the levels of general chromosomal aberrations in H2AX-deficient B cells were at least as high as those observed in concanavalin A-activated H2AX−/− T lymphocytes assayed in parallel (Bassing et al., 2003,Celeste et al., 2003a) (Figures S2 A and S2B, Table 1). Finally, we sequentially analyzed these metaphase spreads via T-FISH (red signal) and then via FISH with a BAC (3′Igh BAC) that specifically recognizes sequences just beyond the most downstream CH (Cα) gene (green signal) and thus would not be altered or deleted by CSR (Figure 1A). These analyses revealed that approximately one-third of all chromosome, but not chromatid, breaks detected by T-FISH in H2AX−/− B cells had a 3′Igh locus BAC signal near the breakpoint (Figures 1B–1D, Table S1 ), suggesting that a sizable percentage of chromosome breaks in H2AX−/− B cells occurred within the IgH locus.
Table 1 |
Genotype | Number of Mice | Mitogen | Number of Metaphases Analyzed | Number of Metaphases with Aberrations (%) | Number of Cytogenetic Aberrations (%) | |||||
Number of Chromosome Breaks | Number of Chromatid Breaks | Number of Other Aberrations | Number of Total Aberrations (%) | |||||||
3 | α-CD40/IL-4 | 90 | 2 (2.2%) | 2 | 0 | 0 | 2 (2.2%) | |||
3 | α-CD40 | 90 | 7 (7.8%) | 3 | 0 | 4 | 7 (7.8%) | |||
3 | Con A | 90 | 1 (1.1%) | 1 | 0 | 0 | 1 (1.1%) | |||
3 | α-CD40/IL-4 | 90 | 2 (2.2%) | 2 | 0 | 0 | 2 (2.2%) | |||
2 | α-CD40 | 60 | 2 (3.3%) | 2 | 1 | 0 | 3 (5%) | |||
3 | Con A | 91 | (7.6%) | 5 | 8 | 1 | 14 (15.2%) | |||
3 | α-CD40/IL-4 | 130 | 50 (36.9%) | 32 | 24 | 21 | 77 (61.2%) | |||
1 | α-CD40 | 30 | 10 (33.3%) | 6 | 9 | 2 | 17 (56.7%) | |||
3 | Con A | 90 | 31(23.3%) | 23 | 9 | 9 | 41 (31.5%) | |||
2 | α-CD40/IL-4 | 100 | 2 (2%) | 2 | 1 | 0 | 3 (3%) | |||
2 | α-CD40/IL-4 | 100 | 0 (0%) | 0 | 0 | 0 | 0 (0%) | |||
2 | α-CD40/IL-4 | 100 | 32 (32%) | 28 | 6 | 2 | 36 (32%) | |||
2 | α-CD40/IL-4 | 100 | 22 (22%) | 17 | 9 | 2 | 28 (28%) | |||
H2AX−/−/p53−/− | 2 | LPS | 60 | 2 (3.3%) | 2 | 0 | 0 | 2 (3.3%) | ||
2 | α-CD40/IL-4 | 60 | 4 (6.7%) | 4 | 1 | 0 | 5 (8.3%) | |||
2 | α-CD40 | 60 | 1 (1.7%) | 1 | 0 | 0 | 1 (1.7%) | |||
2 | Con A | 60 | 1 (1.7%) | 1 | 0 | 0 | 1 (1.7%) | |||
H2AX−/−/p53−/− | 2 | LPS | 60 | 16 (26.7%) | 15 | 3 | 5 | 23 (38.3%) | ||
2 | α-CD40/IL-4 | 60 | 28 (46.7%) | 27 | 9 | 6 | 42 (70.0%) | |||
2 | α-CD40 | 60 | 18 (30%) | 11 | 9 | 10 | 30 (50%) | |||
2 | Con A | 60 | 17 (28.3%) | 13 | 5 | 6 | 24 (40.0%) | |||
2 | α-CD40/Il-4 | 80 | 4 (5.0%) | 3 | 0 | 1 | 4 (5.0%) | |||
2 | α-CD40 | 80 | 1 (1.2%) | 1 | 0 | 0 | 1 (1.2%) | |||
5 | α-CD40/Il-4 | 170 | 45 (26.5%) | 43 | 6 | 8 | 57 (33.5%) | |||
5 | α-CD40 | 170 | 8 (4.7%) | 8 | 2 | 0 | 10 (5.9%) | |||
2 | α-CD40/Il-4 | 60 | 4 (6.6%) | 2 | 2 | 0 | 4 (6.6%) | |||
2 | α-CD40 | 60 | 3 (5%) | 1 | 1 | 1 | 3 (5%) | |||
2 | Con A | 60 | 1 (1.6%) | 0 | 0 | 1 | 1 (1.6%) | |||
2 | α-CD40/Il-4 | 60 | 30 (50%) | 41 | 8 | 6 | 55 (91%) | |||
2 | α-CD40 | 60 | 14 (23.2) | 21 | 6 | 1 | 28 (46.6%) | |||
2 | Con A | 60 | 17 (28.3%) | 16 | 4 | 4 | 24 (40%) | |||
Metaphases of B and T cells activated with the indicated mitogens were hybridized with a telomere probe, counterstained with DAPI, and analyzed for chromosome aberrations. |
Frequent Chromosome Breaks at Igh in H2AX−/− B Cells Activated for CSR
To firmly establish and quantify apparent Igh chromosome breaks in activated H2AX−/− B cells, we performed two-color FISH with the same 3′Igh BAC (labeled for red signal), plus a second BAC (labeled for green signal) that spans the region immediately telomeric to the VH locus (termed 5′Igh BAC) (Figure 2A). The 5′IgH BAC lies just 5′ of the most telomeric VH segment of the Igh locus and, therefore, is not deleted or otherwise altered by V(D)J recombination. In normal cells, both copies of chromosome 12 show red and green signals in the telomeric region (diagrammed as "Intact Igh"; Figure 2A; Table 2). These signals are visualized as two sets of dots when the chromatids are well separated and sometimes as a single set of dots when they are not well separated (diagrammed in Figures 2B and 2C, respectively). Chromosome 12 breaks within the IgH locus result in liberation of a small telomeric portion of chromosome 12, containing the 5′Igh BAC, from the body of chromosome 12. Moreover, in such IgH locus breaks, the 3′Igh BAC will lie very near the breakpoint (Figure 2A; diagrammed as "Chromosome break at Igh"). In such a situation, the small telomeric fragment could be present elsewhere in the metaphase or lost; in either case, we refer to a metaphase harboring a chromosome with a 3′Igh signal and lacking an adjacent 5′Igh signal as having a "split Igh BAC signal," which represents our readout for a break within Igh.
Table 2 |
Genotype | Time in Culture | Percent Metaphases with Igh Breaks (%1 chrom 12/%2 chrom12) | |||||
LPS | α-CD40/ IL-4 | α-CD40 | Con A | ||||
Control Mice | |||||||
Wild-Type | 72 hr | 1 (1/0) | 0 (0/0) | n.d. | 0 (0/0) | ||
H2AX+/− | 72 hr | 0 (0/0) | 1 (1/0) | n.d. | 1 (1/0) | ||
p53−/− | 72 hr | 0 (0/0) | 1 (1/0) | 0 (0/0) | n.d. | ||
AID−/− | 72 hr | 0 (0/0) | 0 (0/0) | n.d. | n.d. | ||
AID−/− | 72 hr | 0 (0/0) | 0 (0/0) | n.d. | n.d. | ||
Mice Deficient for DSB Response Factors | |||||||
H2AX−/− | 72 hr | 2 (2/0) | 9 (6/3) | n.d. | 0 (0/0) | ||
H2AX−/− | 72 hr | 7 (7/0) | 9 (8/1) | 3 (3/0) | 0 (0/0) | ||
H2AX−/− | 72 hr | 3 (3/0) | 10 (10/0) | n.d. | n.d. | ||
H2AX−/− | 72 hr | 7 (7/0) | 20 (18/1) | n.d. | n.d. | ||
H2AX−/− | 72 hr | 6 (5/1) | 10 (8/2) | 0 (0/0) | n.d. | ||
H2AX−/− | 72 hr | 2 (2/0) | 7 (6/1) | 0 (0/0) | n.d. | ||
H2AX−/− | 96 hr | 10 (8/2) | 16 (16/0) | 0 (0/0) | n.d. | ||
H2AX−/− | 96 hr | 8 (8/0) | 21 (17/4) | 3 (3/0) | n.d. | ||
H2AX−/−/p53−/− | 72 hr | 4 (3/1) | 17 (16/1) | 2 (2/0) | n.d. | ||
H2AX−/−/p53−/− | 96 hr | 10 (9/1) | 31 (24/7) | 2 (2/0) | n.d. | ||
H2AX−/−/AID−/− | 72 hr | 0 (0/0) | 0 (0/0) | n.d. | n.d. | ||
H2AX−/−/AID−/− | 72 hr | 0 (0/0) | 0 (0/0) | n.d. | n.d. | ||
53BP1−/− | 72 hr | 2 (2/0) | 10 (7/3) | 1 (1/0) | n.d. | ||
53BP1−/− | 96 hr | 8 (6/2) | 26 (24/2) | 1 (1/0) | n.d. | ||
53BP1−/− | 96 hr | 6 (4/2) | 22 (19/3) | 4 (4/0) | n.d. | ||
53BP1−/− | 96 hr | 1 (1/0) | 10 (10/0) | 0 (0/0) | n.d. | ||
53BP1−/− | 96 hr | 2 (2/0) | 5 (4/1) | 1 (1/1) | n.d. | ||
ATM−/− | 96 hr | 16 (13/3) | 22 (19/3) | 8 (7/1)a | n.d. | ||
ATM−/− | 96 hr | 10 (9/1) | 21 (15/6) | 10 (9/1)a | n.d. | ||
MDC1−/− | 72 hr | 6 (6/0) | n.d. | n.d. | n.d. | ||
MDC1−/− | 72 hr | 6 (6/0) | n.d. | n.d. | n.d. | ||
Two-color Igh FISH was performed on metaphase spreads of the indicated genotypes stimulated with the indicated mitogens; each data point represents the frequency of Igh BAC split signals in 100 metaphases. |
a Expression of surface IgG1, IgG2b, and IgG3 above background was observed in these cultures by FACs analysis at 96 hr. |
Strikingly, we detected split Igh BAC signals in about 10% of the α-CD40/IL-4-stimulated H2AX−/− B cell metaphases after 72 hr stimulation and in about 20% after 96 hr stimulation, a time when more B cells have undergone CSR (Figures 2B–2E, Table 2). We also detected split BAC signals in up to 10% of LPS-stimulated H2AX−/− B cell metaphases (Table 2). As controls, we rarely detected split Igh BAC signals in activated wt or H2AX+/− B cell metaphases and failed to detect any in activated H2AX−/− T cells, despite their similar levels of overall genomic instability (Table 1,Table 2). Moreover, nearly two-thirds of the H2AX−/− B cell metaphases with a split Igh BAC signal retained a small fragment that harbored the 5′Igh sequences, presumably the liberated telomeric fragment of chromosome 12 (e.g., Figures 2B and 2C). Attesting to the sensitivity and specificity of the assay, other than these small fragments, we essentially never observed any chromosomes that harbored the 5′Igh BAC in the absence of the 3′Igh BAC in either mutant or control samples. CSR often occurs on both allelic chromosomes (Dudley et al., 2005); correspondingly, a substantial percentage (15%) of activated H2AX−/− B cells with split Igh BAC signals showed split signals on both chromosomes (Figure 2C and data not shown). This data is in accord with the T- FISH findings, which also showed frequent metaphases with two chromosomes harboring the 3′Igh BAC on their long arms in the absence of a telomere signal (Figure 1C). Finally, as suggested by T-FISH (see above), split Igh BAC signals were associated exclusively with chromosome and not chromatid breaks (Figure 2 and data not shown), in marked contrast to general chromosomal aberrations, which occurred as both chromosome and chromatid breaks (Table 1). Thus, the DNA lesions that led to the Igh chromosomal breaks likely occurred primarily in a prereplicative stage of the cell cycle, in agreement with previous conclusions based on use of γ-H2AX foci as a marker for Igh DSBs (Petersen et al., 2001). We conclude that a large proportion of H2AX−/− B cells activated to undergo CSR generate lesions that result in unrepaired breaks within the IgH locus, potentially contributing to their CSR defect.
Chromosome Breaks at Igh Result from AID Activity
To more directly test whether IgH locus chromosome 12 breaks in activated H2AX−/− B cells resulted from attempted CSR, we took two different approaches. First, we tested H2AX−/− B cells activated with α-CD40 in the absence of IL-4 (or other cytokines), which induces substantial proliferation but generally induces only low-level CSR (Rush et al., 2005). In these experiments, we found a similar high frequency of general chromosomal aberrations in H2AX−/− B cells treated with α-CD40 alone as in those treated with α-CD40 plus IL-4 (Figure 3A, Table 1) but found very few IgH locus-specific DSBs with α-CD40 treatment alone (Figure 3B, Table 2). Second, we generated H2AX−/−/AID−/− B cells by breeding H2AX+/− and AID+/− (Muramatsu et al., 2000) mice. AID deficiency does not affect B cell activation or induction of S region transcription but completely blocks CSR (Muramatsu et al., 2000) (Figure 3C), most likely by eliminating ability to generate S region DSBs (Catalan et al., 2003,Rush et al., 2004,Schrader et al., 2005,Wuerffel et al., 1997). Strikingly, while H2AX−/−/AID−/− B cells showed similar numbers and types of general chromosomal aberrations as H2AX−/− B cells (Figure 3D, Table 1), they lacked detectable IgH locus-specific chromosome 12 breaks at the resolution of our assay (Figure 3E, Table 2). Therefore, the vast majority of Igh chromosomal breaks in activated H2AX−/− B cells depend on activation treatments that induce CSR and are strictly dependent on AID. The fact that these Igh chromosomal breaks are strictly AID dependent, coupled with the additional fact that B cells activated in culture do not undergo somatic hypermutation of variable region exons (the other known AID-dependent process), strongly implies that they were initiated within S regions via attempted CSR.
AID-Dependent Chromosomal Igh Breaks Are Frequent Participants in Translocations
In many human and mouse B cell lymphomas, oncogenes are activated via fusion to the IgH locus, with S regions at translocation junctions (Kuppers and Dalla-Favera, 2001). Strikingly, chromosome 12 translocations that contained the 3′Igh BAC occurred in over 20% of activated H2AX−/− B cell metaphases that contained split Igh BAC signals (Figures 4A–4G and Figure 4I, left bar). Dicentric chromosomes, which can lead to escalating genomic instability via breakage-fusion-bridge (BFB) cycles (Mills et al., 2003), represented about 70% of all translocations in activated H2AX−/− B cells (Figures 4A–4C; Figure 4G; Figure 4J, left bar). In addition, two-color IgH locus FISH combined with chromosome-specific paints revealed that dicentric chromosome 12 derivatives containing the 3′ but not 5′ region of the IgH locus represented a substantial proportion of dicentrics in H2AX−/− B cells (Figure 4G, right panels, and data not shown), likely promoted by the frequent breakage of both chromosome 12s at Igh during attempted CSR (Figures 1C and 2C). Finally, these analyses also revealed translocations between chromosome 12 and other chromosomes in which we could clearly locate the split 3′Igh BAC signal at or near the translocation junction (e.g., Figure 4G, left panels). Based on mouse B cell lymphoma models, one study concluded that S region translocations to c-myc were strictly AID dependent (Ramiro et al., 2004), while another suggested they could be AID independent (Unniraman et al., 2004). Notably, we did not detect Igh translocations in H2AX−/−/AID−/− B cells, despite their substantial level of general genomic instability, clearly demonstrating that the vast majority of Igh translocations in H2AX−/− B cells are AID dependent. We further conclude that the frequent Igh translocations in H2AX−/− B cells are unlikely to involve selection, since they are nonclonal and likely occurred downstream of AID-induced DSBs at S regions, which occur only after a few division cycles subsequent to B cell activation (Rush et al., 2005).
The Tumor Suppressor p53 Exerts Its Function Downstream of the Initial Translocation Event
Prior studies suggested that p53 deficiency may be required to observe chromosomal instability in H2AX-deficient B cells activated for CSR (Celeste et al., 2003a,Reina-San-Martin et al., 2003). However, our current results document dramatic chromosomal instability in B cells deficient for H2AX alone. To assay for any marked effects of p53 deficiency on genomic instability in activated B cells, we compared general and Igh instability in H2AX+/+/p53−/−, H2AX−/−/p53−/−, and H2AX−/−/p53+/+ B cells. Notably, H2AX+/+/p53−/− B cells, which are impaired for multiple checkpoints (Iliakis et al., 2003), appeared similar to wt controls in lacking chromosome 12 breaks or other types of genomic instability (Figure 4H, Table 1). Moreover, H2AX−/−/p53−/− B cells did not show markedly increased genomic instability with respect to frequency or type of aberrations as compared to H2AX−/− B cells (Table 1,Table 2, Figures 4H–4J). Thus, as p53 deficiency does not cause detectable genomic instability in B cells activated for CSR, potential checkpoint deficiencies per se do not appear to account for general or IgH locus genomic instability in activated H2AX−/− cells. While the defective G2/M response to low-level DNA damage associated with H2AX deficiency (Fernandez-Capetillo et al., 2002) conceivably could augment ability to observe broken IgH loci at metaphase, we note that chromosome breaks persist into metaphase in NHEJ-deficient mouse embryonic fibroblasts that have no checkpoint defects (Sekiguchi et al., 2001). We conclude that p53 deficiency is not required for the appearance of dramatic genomic instability, including frequent chromosome breaks and translocations at Igh, in H2AX-deficient B cells activated for CSR in culture.
Increased IgH Locus Chromosomal Breaks and Translocations in 53BP1-, MDC1-, and ATM-Deficient B Cells
We reasoned that factors that interact with γ-H2AX in the context of DSBs and which are required for normal CSR also might function to prevent AID-dependent IgH locus DSBs from progressing to chromosomal breaks. To explore this possibility, we performed two-color Igh FISH on activated B cells from 53BP1-, ATM-, and MDC1-deficient mice. Strikingly, similar to H2AX−/− B cells, a substantial proportion (up to 20% or more) of metaphases from LPS- or α-CD40/IL-4-stimulated ATM−/− and 53BP1−/− B cells harbored Igh split BAC signals; in addition, levels of Igh split BAC signals were increased in α-CD40/IL-4-stimulated cells versus those treated with α-CD40 alone (Table 2). Likewise, in a more limited set of experiments, about 6% of metaphases from LPS-activated MDC1-deficient B cells showed Igh split BAC signals (Table 2). We note that all of these results are highly significant, as IgH locus breaks in similarly stimulated wt cells were extremely rare, as assayed either by the two-color Igh FISH or T-FISH (Table 2). We also characterized, via T-FISH, general chromosomal instability in 53BP1-, ATM-, or MDC1-deficient B cells activated for CSR. Significantly, LPS- and α-CD40/IL-4-activated B cells of all three genotypes displayed dramatically increased levels of chromosomal aberrations compared to wt littermate controls (Table 1). However, while the overall level of chromosomal aberrations in ATM−/− B cells was much greater than that contributed by Igh instability alone, the overall level in activated 53BP1−/− B cells mostly could be attributed to Igh chromosomal breaks (Table 1,Table 2). Correspondingly, α-CD40-stimulated 53BP1−/− B cells showed relatively low levels of chromosomal aberrations compared to H2AX−/− or ATM−/− B cells (Table 1), in accord with findings that 53BP1−/− T cells show limited chromosomal instability (Morales et al., 2006). We employed two-color FISH to analyze metaphases from activated ATM−/− and 53BP1−/− B cells for translocations involving the IgH locus. As for activated H2AX−/− B cells, we found a significant proportion (about 25%) of Igh split BAC signals in activated ATM−/− B cell metaphases in the context of translocations, with a still substantial, but somewhat lower, percentage (13%) in activated 53BP1-deficient B cells (Figure 5A), indicating that a substantial proportion of broken IgH loci enter into chromosomal translocations in all three mutant backgrounds. Moreover, dicentric chromosomes containing split Igh BAC signals were abundant, occurring in a similarly high proportion (about 15%) of activated ATM−/− B cell metaphases as in those of activated H2AX−/− B cells and in about 5% of activated 53BP1−/− B cell metaphases (Figure 5B). To identify Igh translocation partners, we performed SKY on activated ATM−/− B cell metaphases. Analysis of 20 dicentrics revealed that 11 involved chromosome 12 and that about half of those (five of 11) were chromosome 12 to chromosome 12 dicentrics (Table S2 , Figure 5C). In the limited samples analyzed, non-12 partners represented various other chromosomes (Table S2 ). Finally, reprobing these same metaphases with 3′Igh and 5′Igh BACs revealed split Igh BAC signals in most translocations involving chromosome 12 (Figure 5C, Table S2 ). We conclude that, like H2AX−/− B cells, ATM−/− or 53BP1−/− B cells activated for CSR frequently generate chromosomal breaks at the IgH locus that lead to translocations.
H2AX Promotes End Joining in the Context of CSR
We demonstrate that mammalian H2AX prevents AID-dependent DNA breaks in the IgH locus from progressing into chromosome breaks in primary B lymphocytes activated for CSR. During CSR, multiple AID-dependent DSBs may occur within Sμ, some of which can be joined to each other to yield internal Sμ deletions, rather than being joined to downstream S region DSBs to yield CSR (Dudley et al., 2002,Reina-San-Martin et al., 2003). Based on normal internal Sμ deletions in H2AX−/− B cells, prior studies suggested that their CSR defects might be associated with impaired long-range synapses of two different S regions (Reina-San-Martin et al., 2003). While our studies do not address this postulated role, we find that AID-dependent Igh chromosome breaks occur in a large enough proportion of CSR-activated H2AX-deficient B cells to contribute to their CSR defect. We cannot distinguish whether AID-dependent Igh chromosome breaks in H2AX−/− B cells occur during attempted joining of two different S regions, joining of DSBs within a single S region, or both. We note that the residual CSR in H2AX-deficient B cells indicates that end joining is not abrogated. Based on the apparently high level of Sμ internal deletions, DSBs have been proposed to occur more often in Sμ than in downstream S regions, perhaps to drive CSR to downstream S regions (Dudley et al., 2002,Chaudhuri and Alt, 2004). If so, failure to join only a subset of Sμ DSBs could dissociate the V(D)J exon from the CH exons, lead to chromosome breaks, and thereby contribute to defective CSR. The structure of CSR junctions clearly implicates NHEJ and/or a highly related pathway in the process (Dunnick et al., 1993), a notion supported by the dependence of CSR on NHEJ factors (Pan-Hammarstrom et al., 2005,Reina-San-Martin et al., 2003). In this regard, we observe similar IgH locus chromosome breaks in NHEJ-deficient B cells activated for CSR (our unpublished data). Therefore, our studies provide the first direct evidence that mammalian H2AX impacts on this form of DSB repair.
Potential Roles of H2AX in the Joining Phase of CSR
H2AX might influence CSR end joining by several conceivable mechanisms. In yeast, phosphorylated H2A influences NHEJ by altering chromatin structure at DSBs (Downs et al., 2000) and influences HR via a process that brings cohesins to DSBs to tether sister chromatids (van Attikum and Gasser, 2005). In this context, our findings are consistent with an "anchoring" model that proposes that DSB-dependent phosphorylation of H2AX and other repair/checkpoint factors, including 53BP1 and MDC1, by ATM or related PIKK kinases leads to formation of complexes that alter chromatin structure and prevent DSBs from turning into chromosomal breaks, thereby facilitating proper end joining and suppressing translocations (Bassing and Alt, 2004,Rogakou et al., 2000). One prediction of this general model is that DSBs introduced during CSR would turn into chromosome breaks at high frequency in H2AX-deficient cells, and our findings show this to be the case. Another prediction is that DSBs in two different chromosomes in H2AX-deficient cells could often dissociate and be inappropriately joined to cause translocations, a prediction demonstrated by our finding that IgH locus breaks on both copies of chromosome 12 in individual H2AX-deficient B cells often are joined to form dicentric chromosome 12 translocations. A final prediction of this model is that impaired CSR in mice deficient for ATM (Lumsden et al., 2004,Reina-San-Martin et al., 2004), 53BP1 (Manis et al., 2004,Ward et al., 2004), or MDC1 (Lou et al., 2006) would be similarly associated with chromosome 12 breaks and Igh translocations, a prediction that we have confirmed.
The common phenotype of deficiencies for H2AX, ATM, 53BP1, and MDC1 with respect to generation of IgH locus chromosome breaks and translocations in B cells activated for CSR implies that they serve common functions. Given that deficiency for an NHEJ factor shows a similar phenotype (our unpublished data), one common function may involve promotion of NHEJ. However, there are some apparently distinct aspects of the 53BP1-deficient phenotype that are noteworthy. First, while 53BP1 deficiency results in roughly similar levels of Igh chromosomal breaks as H2AX or ATM deficiency, it leads to a nearly complete abrogation of CSR (Manis et al., 2004,Ward et al., 2004). In addition, unlike ATM or H2AX deficiency, 53BP1 deficiency does not lead to dramatic general chromosomal instability, as most observed instability in activated B cells is associated with Igh chromosome breaks. Correspondingly, there is little spontaneous genomic instability in 53BP1-deficient MEFs or T cells (Morales et al., 2003,Morales et al., 2006,Ward et al., 2003b). Thus, these findings are consistent with a more specialized role of 53BP1 in CSR-associated DSBs and/or more effective elimination of cells with other types of DSBs in the 53BP1-deficient versus ATM- or H2AX-deficient backgrounds, with the latter possibility being supported by an increased chromosomal instability in T cells deficient in both 53BP1 and p53 (Morales et al., 2006). However, it also is conceivable that 53BP1 has additional roles in CSR, perhaps upstream of the DSB, via its ability to sense chromatin structural changes (Huyen et al., 2004). Finally, if more extensive studies confirm that MDC1 has as great an effect on IgH locus chromosomal stability as deficiencies for the other factors, its requirement for propagating H2AX phosphorylation by ATM and allowing extended focus formation (Lou et al., 2006) might suggest that putative tethering functions of H2AX would occur by more short-range mechanisms.
Does H2AX Promote End Joining Beyond CSR?
We have been able to follow AID-dependent Igh DSBs because they occur at high frequency. Thus, the question arises as to how applicable our findings are to general DSBs. In this regard, H2AX-deficient B cells, T cells, MEFs, and embryonic stem cells all show marked increases in general genomic instability including chromosome breaks and translocations. However, while the IgH locus chromosome breaks associated with CSR appear to be initiated in a prereplicative cell cycle phase, those that lead to general instability, at least as observed in H2AX-deficient B cells, appear to occur over a broader portion of the cell cycle. On the other hand, while V(D)J recombination employs a DSB, there has been no measured effect of H2AX deficiency on this process (Bassing et al., 2002,Celeste et al., 2002). In this context, the ability of RAG to hold ends together in a postcleavage synaptic complex and coordinate their repair via NHEJ (Gellert, 2002) might largely prevent RAG-induced DSBs from progressing into chromosomal breaks in H2AX-deficient cells, although H2AX may function in further stabilizing such DSBs (Bassing and Alt, 2004). Support for an H2AX role in V(D)J recombination comes from the finding that H2AX/p53-deficient mice often develop pro-B lymphomas with complicons resulting from translocations that fuse the IgH JH locus to sequences downstream of c-myc (Bassing et al., 2003,Celeste et al., 2003a) and that these H2AX/p53-deficient complicons are similar in structure to RAG-dependent complicons in NHEJ/p53-deficient mice (Bassing and Alt, 2004). Finally, it remains possible that subtle defects in joining of RAG-initiated DSBs could contribute to the lower lymphocyte numbers in H2AX−/− mice (Celeste et al., 2002).
P53 Functions Downstream of Translocations to Suppress H2AX-Deficient B Lymphomas
We show that H2AX deficiency per se is sufficient to generate a large pool of activated B cells that harbor AID-dependent chromosomal translocations involving the IgH locus. Yet, H2AX-deficient mice are not markedly cancer prone and, in particular, have not been found to be susceptible to B cell lymphomas. On the other hand, H2AX−/−p53−/− mice are dramatically cancer prone, and most characterized H2AX−/−p53−/− tumors have clonal translocations, including B lineage lymphomas that harbor Igh translocations that lead to c-myc activation (Celeste et al., 2003a,Bassing et al., 2003). Together with our current results, the latter finding supports the notion that defective checkpoints associated with p53 deficiency may not be required for generation of potentially oncogenic translocations, but rather to allow expansion of cells harboring them. For example, p53 deficiency may permit cells to continue cycling in the presence of chromosome breaks and other forms of genomic instability (Mills et al., 2003) and/or allow activated oncogene (e.g., c-myc) expression from translocations without inducing apoptosis (Hemann et al., 2005).
Experimental Procedures
We used previously described H2AX-deficient mice (Bassing et al., 2003), 53BP1 (Morales et al., 2003), ATM (Borghesani et al., 2000), and MDC1 (Lou et al., 2006). Mice doubly deficient for H2AX and p53 (Donehower et al., 1992) or for H2AX and AID (Muramatsu et al., 2000) and their control littermates were generated by breeding.
Mature B and T Cell Purification and Cultures
Splenocyte single-cell suspensions were prepared and fractionated into CD43+ and CD43− populations as described (Manis et al., 2004). For B cell stimulation, the CD43− fraction was incubated with the B cell mitogens bacterial lypopolysaccharide (LPS, 20 μg/mL; Sigma), anti-CD40 antibody (α-CD40, 1 μg/mL, BD Pharmingen) and interleukin 4 (IL-4, 20 ng/mL, R&D Systems). For T cell stimulation, the CD43+ fraction was incubated with concanavalin A (2.5 μg/mL, Sigma).
Assessment of CSR by Flow Cytometry
Stimulated B cells (0.5 million) were washed with 2% FCS/PBS; stained with antibodies labeled with Cy-Chrome (B220), fluorescein (IgG2b, IgG1), or phycoerithrin (IgE and IgG3) for 15 min; and analyzed in a FACSCAN using Cellquest (Becton Dickinson) or Flo-Jo (Tree Star) software. At least 10,000 events of live lymphoid cells were recorded.
FISH Probes
We detected the 3′ end of the IgH locus using BAC199 (a gift of Barbara Birshtein, Albert Einstein College of Medicine, New York) and the 5′ end using BAC207 (a gift of Peter Brodeur, Tufts University, Boston). FITC-conjugated paints for mouse chromosomes 12 were purchased from Applied Spectral Imaging. For telomere staining, a Cy3-conjugated (TTAGGG)3 PNA probe was purchased from Applied Biosystems.
PNA FISH of TTAGGG Sequences
Lymphocytes were incubated in colcemid (KaryoMAX, GIBCO), swollen in prewarmed 30 mM sodium citrate for 25 min at 37°C, fixed in methanol/acetic acid (3/1), and air dried on slides overnight. After pepsin digestion (1 mg/ml, 10 min at 37°C), slides were denatured at 80°C for 3 min, hybridized with a Cy3-labeled PNA telomeric probe (Cy3-[TTAGGG]3) in 70% formamide at RT for 2 hr, washed, dehydrated, and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, California). Metaphase images were captured using a Nikon Eclipse microscope equipped with a CCD camera (Applied Spectral Imaging, Carlsbad, California) and a 63× objective lens. For cytogenetic analysis, at least 30 metaphases of each genotype were scored for chromosomal aberrations. After image recording, selected slides were washed in 2XSSC, dehydrated through serial ethanols, and reprobed with BACs as described below.
Two-Color Fluorescence In Situ Hybridization
BACs were labeled with either biotine (Biotin-Nick Translation Mix, Roche) or digoxigenin (Dig-Nick Translation Mix, Roche), as per manufacturer's instructions. Two hundred nanograms of BAC DNA was precipitated with mouse Cot1 DNA (Invitrogen; ratio of BAC DNA to Cot1 DNA, 1:20), resuspended in 15 μl of hybridization solution (50% formamide, 2X SSC, 10% dextran sulfate, 0.15% SDS), and codenatured on slides for 5 min at 76°C. Slides were then incubated at 37°C for 16 hr, washed in 50% formamide/2X SSC for 5 min twice at 45°C and 2X SSC for 5 min twice at 45°C, and incubated with avidin-Cy3 and antidigoxigenin-FITC (Roche, 1:250 dilution) in 2XSSC/0.05% Tween-20 for 1 hr at RT. After three washes in 2XSSC/0.05% Tween-20, slides were mounted in Vectashield with DAPI (Vector Laboratories). Metaphase images were captured using a Nikon Eclipse microscope equipped with a CCD camera (Applied Spectral Imaging, Carlsbad, California) and a 63× objective lens. One hundred metaphases were analyzed for each sample. After image recording, selected slides were dehydrated through serial ethanols and reprobed with fluorochrome-conjugated paints to mouse chromosome 12, following manufacturer's instructions.
Spectral Karyotype Analysis
Metaphase spreads were hybridized using a SkyPaint DNA Kit (Applied Spectral Imaging), following manufacturer's instructions. Spectral images were captured and analyzed using an interferometer and software from Applied Spectral Imaging. Selected slides were dehydrated through serial ethanols, and two-color FISH was performed as described above.
We thank Charles Lee, Hwei-Ling Cheng, Tiffany Borjeson, and Alyssa Riley for technical advice; Barbara Haskell for technical support; and all Alt lab members for helpful discussions. This work was supported by NIH Grants PO1CA092625-05 and 2PO1AI031541-15 (to F.W.A.), GM65812 (to P.B.C.), and RO1 CA89239 and CA92312 (to J.C.); a Long-Term Fellowship of the European Molecular Biology Organization (to S.F.); a Fellowship of the Leukemia and Lymphoma Society (to S.Z.); and a NIA/NIHKO8 Award (to D.B.L.). Z.L. is the recipient of a DOD Breast Cancer Postdoctoral Fellowship (DAMD17-03-1-0610). C.H.B. was a Lymphoma Research Foundation Fellow and is a Pew Scholar in the Biomedical Sciences. J.C. is a recipient of DOD Breast Cancer Career Development Award (DAMD17-02-1-0472). F.W.A. is a Howard Hughes Medical Institute investigator.
Bassing and Alt, 2004 (2004). H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity. Cell Cycle 3, 149–153. CrossRef | PubMed
Bassing et al., 2002 (2002). Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc. Natl. Acad. Sci. USA 99, 8173–8178. CrossRef | PubMed
Bassing et al., 2003 (2003). Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors. Cell 114, 359–370. Abstract | Full Text | PDF (547 kb) | CrossRef | PubMed
Bekker-Jensen et al., 2005 (2005). Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1. J. Cell Biol. 170, 201–211. CrossRef | PubMed
Borghesani et al., 2000 (2000). Abnormal development of Purkinje cells and lymphocytes in Atm mutant mice. Proc. Natl. Acad. Sci. USA 97, 3336–3341. CrossRef | PubMed
Catalan et al., 2003 (2003). The block in immunoglobulin class switch recombination caused by activation-induced cytidine deaminase deficiency occurs prior to the generation of DNA double strand breaks in switch mu region. J. Immunol. 171, 2504–2509. PubMed
Celeste et al., 2002 (2002). Genomic instability in mice lacking histone H2AX. Science 296, 922–927. CrossRef | PubMed
Celeste et al., 2003a (2003). H2AX haploinsufficiency modifies genomic stability and tumor susceptibility. Cell 114, 371–383. Abstract | Full Text | PDF (900 kb) | CrossRef | PubMed
Celeste et al., 2003b (2003). Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat. Cell Biol. 5, 675–679. CrossRef | PubMed
Chaudhuri and Alt, 2004 (2004). Class-switch recombination: interplay of transcription, DNA deamination and DNA repair. Nat. Rev. Immunol. 4, 541–552. PubMed
Chen et al., 2000 (2000). Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX. Science 290, 1962–1965. CrossRef | PubMed
Donehower et al., 1992 (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221. CrossRef | PubMed
Downs et al., 2000 (2000). A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature 408, 1001–1004. CrossRef | PubMed
Dudley et al., 2002 (2002). Internal IgH class switch region deletions are position-independent and enhanced by AID expression. Proc. Natl. Acad. Sci. USA 99, 9984–9989. CrossRef | PubMed
Dudley et al., 2005 (2005). Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences. Adv. Immunol. 86, 43–112. CrossRef | PubMed
Dunnick et al., 1993 (1993). DNA sequences at immunoglobulin switch region recombination sites. Nucleic Acids Res. 21, 365–372. PubMed
Fernandez-Capetillo et al., 2002 (2002). DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1. Nat. Cell Biol. 4, 993–997. CrossRef | PubMed
Fernandez-Capetillo et al., 2003 (2003). H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis. Dev. Cell 4, 497–508. Abstract | Full Text | PDF (660 kb) | CrossRef | PubMed
Fernandez-Capetillo et al., 2004 (2004). H2AX: the histone guardian of the genome. DNA Repair (Amst.) 3, 959–967. CrossRef | PubMed
Gellert, 2002 (2002). V(D)J recombination: RAG proteins, repair factors, and regulation. Annu. Rev. Biochem. 71, 101–132. CrossRef | PubMed
Hemann et al., 2005 (2005). Evasion of the p53 tumour surveillance network by tumour-derived MYC mutants. Nature 436, 807–811. CrossRef | PubMed
Honjo et al., 2004 (2004). AID: how does it aid antibody diversity?. Immunity 20, 659–668. Abstract | Full Text | PDF (342 kb) | CrossRef | PubMed
Huyen et al., 2004 (2004). Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature 432, 406–411. CrossRef | PubMed
Iliakis et al., 2003 (2003). DNA damage checkpoint control in cells exposed to ionizing radiation. Oncogene 22, 5834–5847. CrossRef | PubMed
Kobayashi et al., 2002 (2002). NBS1 localizes to gamma-H2AX foci through interaction with the FHA/BRCT domain. Curr. Biol. 12, 1846–1851. Abstract | Full Text | PDF (365 kb) | CrossRef | PubMed
Kracker et al., 2005 (2005). Nibrin functions in Ig class-switch recombination. Proc. Natl. Acad. Sci. USA 102, 1584–1589. CrossRef | PubMed
Kuppers and Dalla-Favera, 2001 (2001). Mechanisms of chromosomal translocations in B cell lymphomas. Oncogene 20, 5580–5594. CrossRef | PubMed
Lou et al., 2006 (2006). MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals. Mol. Cell 21, 187–200, this issue. Abstract | Full Text | PDF (1025 kb) | CrossRef | PubMed
Lumsden et al., 2004 (2004). Immunoglobulin class switch recombination is impaired in Atm-deficient mice. J. Exp. Med. 200, 1111–1121. CrossRef | PubMed
Manis et al., 2004 (2004). 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination. Nat. Immunol. 5, 481–487. CrossRef | PubMed
Mills et al., 2003 (2003). The role of DNA breaks in genomic instability and tumorigenesis. Immunol. Rev. 194, 77–95. CrossRef | PubMed
Morales et al., 2003 (2003). Role for the BRCA1 C-terminal repeats (BRCT) protein 53BP1 in maintaining genomic stability. J. Biol. Chem. 278, 14971–14977. CrossRef | PubMed
Morales et al., 2006 (2006). 53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis. Proc. Natl. Acad. Sci. USA , in press. PubMed
Muramatsu et al., 2000 (2000). Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563. Abstract | Full Text | PDF (865 kb) | CrossRef | PubMed
Pan-Hammarstrom et al., 2005 (2005). Impact of DNA ligase IV on nonhomologous end joining pathways during class switch recombination in human cells. J. Exp. Med. 201, 189–194. CrossRef | PubMed
Petersen et al., 2001 (2001). AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching. Nature 414, 660–665. CrossRef | PubMed
Ramiro et al., 2004 (2004). AID is required for c-myc/IgH chromosome translocations in vivo. Cell 118, 431–438. Abstract | Full Text | PDF (327 kb) | CrossRef | PubMed
Reina-San-Martin et al., 2003 (2003). H2AX is required for recombination between immunoglobulin switch regions but not for intra-switch region recombination or somatic hypermutation. J. Exp. Med. 197, 1767–1778. CrossRef | PubMed
Reina-San-Martin et al., 2004 (2004). ATM is required for efficient recombination between immunoglobulin switch regions. J. Exp. Med. 200, 1103–1110. CrossRef | PubMed
Reina-San-Martin et al., 2005 (2005). Genomic instability, endoreduplication, and diminished Ig class-switch recombination in B cells lacking Nbs1. Proc. Natl. Acad. Sci. USA 102, 1590–1595. CrossRef | PubMed
Riblet, 2004 (2004). Immunoglobulin heavy chain genes of mouse. In Molecular Biology of B Cells. Honjo, T.A., Alt, F.W., Neuberger, M., eds. (London: Elsevier Academic Press), pp. 19–26. PubMed
Rogakou et al., 1999 (1999). Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146, 905–916. CrossRef | PubMed
Rogakou et al., 2000 (2000). DNA double-stranded breaks induce histone H2AX phosphorylation of serine 139. J. Biol. Chem. 273, 5858–5868. CrossRef | PubMed
Rush et al., 2004 (2004). Staggered AID-dependent DNA double strand breaks are the predominant DNA lesions targeted to S mu in Ig class switch recombination. Int. Immunol. 16, 549–557. CrossRef | PubMed
Rush et al., 2005 (2005). Expression of activation-induced cytidine deaminase is regulated by cell division, providing a mechanistic basis for division-linked class switch recombination. Proc. Natl. Acad. Sci. USA 102, 13242–13247. CrossRef | PubMed
Schrader et al., 2005 (2005). Inducible DNA breaks in Ig S regions are dependent on AID and UNG. J. Exp. Med. 202, 561–568. CrossRef | PubMed
Sekiguchi et al., 2001 (2001). Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development. Proc. Natl. Acad. Sci. USA 98, 3243–3248. CrossRef | PubMed
Stewart et al., 2003 (2003). MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature 421, 961–966. CrossRef | PubMed
Stucki et al., 2005 (2005). MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell 123, 1213–1226. Abstract | Full Text | PDF (972 kb) | CrossRef | PubMed
Unniraman et al., 2004 (2004). Identification of an AID-independent pathway for chromosomal translocations between the Igh switch region and Myc. Nat. Immunol. 5, 1117–1123. CrossRef | PubMed
van Attikum and Gasser, 2005 (2005). The histone code at DNA breaks: a guide to repair?. Nat. Rev. Mol. Cell Biol. 6, 757–765. CrossRef | PubMed
Vogelstein et al., 2000 (2000). Surfing the p53 network. Nature 408, 307–310. CrossRef | PubMed
Ward et al., 2003a (2003). Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J. Biol. Chem. 278, 19579–19582. CrossRef | PubMed
Ward et al., 2003b (2003). p53 binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice. Mol. Cell. Biol. 23, 2556–2563. CrossRef | PubMed
Ward et al., 2004 (2004). 53BP1 is required for class switch recombination. J. Cell Biol. 165, 459–464. CrossRef | PubMed
Wuerffel et al., 1997 (1997). Ig Sgamma3 DNA-specifc double strand breaks are induced in mitogen-activated B cells and are implicated in switch recombination. J. Immunol. 159, 4139–4144. PubMed
Xie et al., 2004 (2004). Control of sister chromatid recombination by histone H2AX. Mol. Cell 16, 1017–1025. Abstract | Full Text | PDF (472 kb) | CrossRef | PubMed
Publication Information
Received: November 12, 2005
Revised: December 16, 2005
Accepted: January 5, 2006
Published: January 19, 2006


text:
My phone has been ringing off the hook today with last-minute submitters to the Search Engine Watch Awards asking for an extension to today's deadline.
In an effort to get all these great submissions included in our awards, we've decided to extend our deadline through Tuesday, July 21. Besides that, it will allow me to get something done today without answering my phone or e-mail about this every few minutes. :)
To make your submission, or for more information, visit the SEW Awards page.
Anyone that rushed their submission to meet today's deadline and would like to add more to theirs can do so by contacting us and letting us know which submission you'd like to revise.
Thanks again for your interest in the SEW Awards. I hope this extension will help you all get your best submissions in for consideration.
Meet Your Favorite Search Engine Watch Contributors
Many of SEW's leading expert contributors will be at ClickZ Live, the new online and digital marketing event kicking off in New York (March 31-April 3). Hear from the likes of:
Thom Craver,
Josh Braaten,
Lisa Barone,
Simon Heseltine,
Josh McCoy,
Lisa Raehsler,
Greg Jarboe,
Dan Cristo,
Joseph Kerschbaum,
John Gagnon,
Eric Enge and more!

subset: commoncrawl


text:
Based on classic American mixing bowls, these handcrafted earthenware dog bowls have wide openings that provide comfortable access for all sizes and breeds. Decorated with an image of a stick, a wide lip around the rim makes the bowl easy to handle.
This item is shipped from our warehouse by UPS to arrive within five business days of our receiving your order.
The shipping rate varies depending on your order total and shipping destination.View Shipping Options and Charges.
For an additional charge of $15 per address, we can rush delivery to most destinations. This service is not available for some oversize items, items shipped directly from the supplier (including perishable and custom-manufactured items), out-of-stock items and items to be shipped outside the contiguous 48 states.
At Williams-Sonoma, we take great pride in the quality and craftsmanship of our products. If, within 90 days, you are not completely satisfied with your purchase for any reason, please return the item for an exchange or refund of the merchandise value. View Full Return Policy.
Item | Price | Flat-Rate Delivery Fee |
Ridged Dog Bowl, Medium, Tangerine | $29.95 | $0 |
Ridged Dog Bowl, Large, Tangerine | $39.95 | $0 |
Ridged Dog Bowl, Medium, Taupe | $29.95 | $0 |
Ridged Dog Bowl, Large, Taupe | $39.95 | $0 |


text:
We made a Tyson Young Whole Chicken in the oven. The macros for it on the back of the package says:
4 oz
210 cals
15g fat
19g pro
0g carb
Now, that's assuming you grab a random batch of meat as the chicken included thigh, leg, wing, etc.
I only grabbed 8 oz. of the breast meat. Should I just log it in my MyFitnessPal as 8 oz. of Tyson Boneless Skinless Chicken Breast which would be 50g protein, 2g fat? Or should I still log it as the Young Whole Chicken?
Thread: Chicken breast macro help!


text:
Benjamin C. French, PhD
Benjamin C. French, PhD
Assistant Professor of Biostatistics in Biostatistics and Epidemiology at the Hospital of the University of Pennsylvania
Department: Biostatistics and Epidemiology
Graduate Group Affiliations
Contact information
Department of Biostatistics and Epidemiology
University of Pennsylvania Perelman School of Medicine
204 Blockley Hall
423 Guardian Drive
Philadelphia, PA 19104-6021
University of Pennsylvania Perelman School of Medicine
204 Blockley Hall
423 Guardian Drive
Philadelphia, PA 19104-6021
Office: 215-573-8545
Fax: 215-573-4865
Fax: 215-573-4865
BA (Mathematics and Norwegian)
St. Olaf College, Northfield, Minnesota, 2001.
MS (Biostatistics)
University of Washington, Seattle, Washington, 2004.
PhD (Biostatistics)
University of Washington, Seattle, Washington, 2008.
Permanent linkBA (Mathematics and Norwegian)
St. Olaf College, Northfield, Minnesota, 2001.
MS (Biostatistics)
University of Washington, Seattle, Washington, 2004.
PhD (Biostatistics)
University of Washington, Seattle, Washington, 2008.
Selected Publications
Goldberg DS, French B, Forde KA, Groenveld PW, Bittermann T, Backus L, Halpern SD, Kaplan DE: The Downside of Centralized Care: Access to Liver Transplantation in the Veterans Affairs Health System. Journal of the American Medical Association 2014 (in press).Vorovich EE, French B, Ky B, Goldberg L, Fang J, Sweitzer N, Cappola TP.: Biomarker Predictors of Cardiac Hospitalization in Chronic Heart Failure: A Recurrent Event Analysis. American College of Cardiology Scientific Sessions (Oral Presentation) 2014.
Yehia BR, French B, Fleishman JA, Metlay JP, Berry SA, Korthuis PT, Agwu AL, Gebo KA: Retention in Care is More Strongly Associated with Viral Suppression in HIV-Infected Patients with Lower versus Higher CD4 Counts. Journal of Acquired Immune Deficiency Syndromes 65(3): 333-9, 2014.
Sutton RM, Niles D, French B, Maltese MR, Leffelman J, Eilevstjonn J, Wolfe H, Nishisaki A, Meany PA, Berg RA, Nadkarni VM: First Quantitative Analysis of Cardiopulmonary Resuscitation Quality During In-Hospital Cardiac Arrests of Young Children. Resuscitation 85(1): 70-4, 2014.
Zhang KW, French B, May Khan A, Plappert T, Fang JC, Sweitzer NK, Borlaug BA, Chirinos JA, St John Sutton M, Cappola TP, Ky B: Strain Improves Risk Prediction Beyond Ejection Fraction in Chronic Systolic Heart Failure. Journal of the American Heart Association 3(1): e000550, 2014.
Ky B, Putt M, Sawaya H, French B, Januzzi JL, Sebag IA, Plana JC, Cohen V, Banchs J, Carver JR, Wiegers SE, Martin RP, Picard MH, Gerszten RE, Halpern EF, Passeri J, Kuter I, Scherrer-Crosbie M.: Early Increases in Multiple Biomarkers Predict Subsequent Cardiotoxicity in Breast Cancer Patients Treated with Doxorubicin, Taxanes, and Trastuzumab. Journal of the American College of Cardiology 63(3): 809-16, 2014.
French B, Saha-Chaudhuri P, Ky B, Cappola TP, Heagerty PJ: Development and Evaluation of Multi-Marker Risk Scores for Clinical Prognosis. Statistical Methods in Medical Research 2013 (in press).
Topjian AA, French B, Sutton RM, Conlon T, Nadkarni VM, Moler FW, Dean JM, Berg R: Early Post-Resuscitation Hypotension is Associated with Increased Mortality Following Pediatric Cardiac Arrest. Critical Care Medicine 2013 (in press).
McLeod L, French B, Flynn JM, Dormans JP, Keren R: Antifibrinolytic Use and Blood Transfusions in Pediatric Scoliosis Surgeries Performed at US Children's Hospitals. Journal of Spinal Disorders and Techniques 2013 (in press).
Betesh J, VanZandbergen C, Fuchs BD, Tait G, Hanish A, Fishman N, French B, Umscheid CA: The Impact of an Automated Early Warning System for Sepsis. American Medical Informatics Association Annual Symposium (Oral Presentation) 2013.


text:
IEEE-USA Home: Communications: e-Books
The purpose of this report is to describe the requirements for a Federal employee's disclosure of wrongdoing to be legally protected as whistle blowing under current statutes and case law.
------------------------------------------ Member Price: FREE Non-member Price: $4.99
Buy this e-Book
* to receive the Member discount on eligible products, please log in on the next screen with your IEEE Web Account.
Updated: 05 September 2012 Contact: email@example.com
Terms & Conditions - Privacy and Security - Contacts/Info


text:
Montclair parents urged to be healthy eating role models
The Montclair Times
Healthy food |for kids
Registered dietician Jamie Pula offers these suggestions:
• Encourage your child to make a healthy snack with you, and then name it after him or her.
• Stop being a short-order cook. Don't make different dishes just to please your child.
• Reward your children with hugs and kisses instead of snacks. Don't substitute snacks or goodies for meals.
• Use family mealtime to talk about fun and happy things together. Put away cell phones and iPads.
• When your children are hungry between meals, offer healthy choices such as yogurt, fruit, or a raw vegetable.
Contact George Wirt at email@example.com.


text:
Boston Celtics Edge 76ers, Take Game 1
The Boston Celtics came back to defeat the Philadelphia 76ers 92-91 in Game 1 of the Conference Semifinals. The Boston Globe's Dan Shaughnessy and the Philadelphia Daily News' Bob Cooney report.
Today in Philly Sports
Each week, cartoonist Rob Tornoe offers his take on Philly sports and asks you to help finish it. Click here to see this week's cartoon.
Connect with Philly.com Sports
Featured Photo Galleries


text:
I thought I would start this thread to see what your favorite apps are. Being a new user to the iPad community I can honestly say I love the following apps after a day's use (mind you it was a full day's use)
1. Flipboard
2. Zite
3. Zootool (for my iPhone)
Evernote will probably right up there as soon as my classes begin! But I just wanted tonshare how much i love my iPad 2!!!
What are your Top 3 apps?
Posted from my IPad 2


text:
Talks aimed at averting strikes by fuel tanker drivers have been adjourned and will resume on Tuesday.
Leaders of the Unite union spent all day at the conciliation service Acas to try to reach a deal with distribution companies to end a row over a number of issues including pensions and terms and conditions.
Peter Harwood, Acas chief conciliator, said: "Acas talks have adjourned for the day. I am encouraged that the parties are still positively engaged in the process. Talks will reconvene tomorrow morning."
Unite will have to decide on Tuesday whether to call industrial action.
Under employment law the union will have to give seven days' notice of any walkouts.


text:
Results 1 to 3 of 3
Thread: Tire Opinions
- Join Date
- Sep 2011
- Location
- Hermantown, MN
- Posts
- 12
Revo 2 or Terra Grapplers?
Hey everyone. I'm looking at replacing the tires on my '05 1500 Silverado before winter rolls in, which could be tomorrow up here...you never know. I just bought the truck last month and it came with Cooper Discoverer H/T's which I'm not a fan of. I'm looking at either Bridgestone Revo 2's or Nitto Terra Grapplers. I had the original Revo's on an old K1500 Blazer I used to own and loved them, but I haven't heard from anyone that has the 2's. I also don't have any experience with the Terra Grapps, but have read lots of good reviews. Just looking to hear what everyone thinks, pros/cons, rants/raves, etc. Thanks!
Last edited by npete7381; 10-18-2011 at 09:50 PM.
10-18-2011, 10:48 PM #2I've heard that a lot of people really like the nitto's. Haven't heard to much on the bridgestones.
2006 Duramax LBZ
Edge Evo
4'' Diamond eye to 6'' tip
16'' ion alloy, 33'' fierce attitudes
Lots of work left to do!
10-19-2011, 01:23 PM #3I think the Nitto's are more of a A/T tire than the Bridgstones.
2005 Chevy Silverado Z71 Crew Cab
6" Skyjacker lift w/Edlebrock dual front remote resoviour shocks, single rear remote. Rancho skid-plate.
17" x 9" M/T Classic Lock Wheels, 35" BFG A/T or M/T tires
4.10 PercisionGears w/factory locker, T/A Performance rear-end cover
Banks CAI intake, ported throttlebody, JBA coated headers, over-sized Magnaflow cats, Magnaflow cat back exhaust, Diablo tuned
B&M aluminum deep transmission pan
Warn brush guard w/Warn 9.5 XP winch, PIAA bulbs, Line-X bedliner, AWS Black tool chest, Extang tonneau cover
Weather Tech floor mats
Cover-Craft seat covers
Dash mat cover
Formula One window tint
One black Lab in rear seat.
Similar Threads
Goodyear Wrangler Authority Tire - The Secret Tire
By countryboy73703 in forum Wheels & TiresReplies: 51Last Post: 11-07-2013, 04:31 PM -
4 Factory Aluminum 15" Rims and spare tire with tire
By Crawdaddy in forum Wheels & Tires ClassifiedsReplies: 0Last Post: 07-07-2011, 11:47 AM -
XFE opinions
By tnhusker in forum Chevy Silverado Forum (GMC Sierra)Replies: 2Last Post: 12-31-2009, 10:25 PM -
Tire Review - Pathfinder Trail A/P from Discount Tire Company
By Steve in forum Product ReviewsReplies: 0Last Post: 07-20-2009, 06:09 PM -
1999 k1500 ls tire no lift max tire size.....
By ultramegabob in forum Chevy Suburban Forum (GMC Yukon XL)Replies: 2Last Post: 02-18-2007, 12:35 AM
Tags for this Thread


text:
Fall of Reach
Talk236this wiki
Redirected from Battle of Reach
| ||
| ||
| ||
Fall of Reach | ||
| ||
| ||
| ||
| ||
Belligerents | ||
| ||
Commanders | ||
| ||
Strength | ||
| ||
Casualties | ||
| ||
- "We knew this day would come. They have found our fortress among the stars. The Covenant are on Reach. They will burn this planet, kill millions, and when Reach falls—and fall it will—there will be nothing left to stand between them... and Earth."
The Fall of Reach,[6][7] sometimes referred to as the Battle of Reach, was one of the largest engagements in the Human-Covenant war. It was fought between the UNSCDF and the Covenant from July 24th to August 30th 2552, however a small number of human survivors remained on the planet until September 7th.
Despite suffering a severe defeat at the Battle of Sigma Octanus IV, the Covenant managed to place a tracking device on the UNSC Iroquois and used it to find other human colony worlds. When the Iroquois returned to Reach, the human presence - and its extent - was disclosed and later revealed to the Covenant fleet.[8][9] Ironically, the location of Reach was already known to the Covenant; a previously recovered Forerunner artifact indicated Reach held another artifact.
At first, the Covenant sent a pre-invasion force, consisting of a few SDV-class heavy corvettes and a CSO-class supercarrier, the Long Night of Solace, to Reach and secretly deployed a large force in the Viery Territory. This invasion force was cloaked from UNSC sensors by sophisticated Stealth pylons deployed around it. The Covenant also sent a small Sangheili Zealot strike team led by a Field Marshall to recover a Forerunner artifact from an excavation site near the Visegrád communications outpost.
Winter ContingencyEdit
- "Colonel, this is Noble One. There are no rebels, the Covenant are on Reach."—Carter-A259[10]
Before the discovery of the Covenant invasion force, Noble Team was sent to investigate a signal blackout at the communications array outpost that kept Reach in contact to the rest of the UNSC. Sabotage by Insurrectionists was suspected. As Noble Team arrived at the station, they discovered that the Covenant was behind the disturbance. After rescuing pinned-down UNSC troops and finding the remaining survivors at the station, Noble Team was ambushed by an elite team of Covenant Zealots. The only Zealot that escaped was the leader of the team, an Elite Field Marshall.[11]
Opening skirmishesEdit
- "Winter Contingency has been declared, all units are mobilized and ready."—UNSC Officer
The Covenant created a beachhead on Reach in the Viery Territory with anti-air batteries surrounding the perimeter of their staging area. With several Spires, the Covenant covertly inserted massive invasion armies, including several Corvettes, without detection. After the skirmishes at the communications array, Covenant forces attacked Sword Base in an attempt to overrun the ONI facility and possibly even find the forerunner artifacts under the facility. However, these forces were repulsed after Noble Team reinforced the base and brought its anti-air turrets and communications back online. The Covenant Corvette leading the attack was forced to retreat and was destroyed by an orbital MAC round.[12]
With reports of Covenant deployments on Reach becoming ever-more frequent, the UNSC decided to initiate a reconnaissance operation in order to ascertain what the Covenant were hiding in the "Dark Zone". Upon discovering that the Covenant were massing a large invasion force, the UNSC organised a counter-offensive.[13]
UNSC counterattackEdit
- "…we are now under attack across the Viery territory, including Orbital Defences. As per the Winter Contingency, we are countering on every front."
- Main articles: Battle of Viery and Operation: UPPERCUT
As the fighting across the Viery territory escalated, the UNSC Army launched it's offensive at the Covenant's staging ground in an attempt to overrun the Covenant forces. The assault was spearheaded by Noble Team. Throughout the assault, members of Noble Team destroyed several of the Covenant anti-air batteries enabling the UNSC to achieve air superiority. Supported by Longswords and Frigates, the UNSC Army continued it's drive across the Covenant staging areas. However, the situation changed when the CSO-class supercarrier, Long Night of Solace, appeared over the area and reinforced the Covenant forces. As a result, many UNSC frigates were lost and the assault ground to a halt.[14]
As the Covenant grew in number and in strength, the UNSC began to lose their initial home-field advantage. On the ground, the UNSC would never successfully go on the offensive again and although they would hold off the Covenant in certain areas such as New Alexandria, any surviving UNSC forces were ultimately glassed.
One of the first defeats suffered by the UNSC on Reach was the Covenant's second attack on Sword Base, this time actually glassing areas around the Base. The arrival of the supercarrier led to Operation: UPPERCUT, where Noble Team used Sabres and the corvette Ardent Prayer to deliver a "Slipspace Bomb" to the supercarrier and destroy it. Unfortunately, during the battle, the Slipspace drive malfunctioned and wasn't able to detonate remotely. Jorge-052 voluntarily sacrificed himself to manually trigger the drive. Just as the supercarrier was destroyed, more Covenant ships arrived, this time in the form of Cruisers and Assault Carriers, to reinforce the Covenant forces already present on Reach.[15]
Further engagementsEdit
- Main articles: Raid of New Alexandria and Evacuation of New Alexandria
Over the next week, Covenant ground forces landed on the planet and Covenant ships began glassing targeted areas. A battle broke out in the city of New Alexandria, with large numbers of civilian evacuation transports being shot down by Covenant air and ground forces. Units from Noble Team assisted Army and Marine forces in clearing evacuation zones, succeeding in damaging a Covenant corvette in the process. Though UNSC forces, with great sacrifice, ultimately succeeded in clearing the majority of Covenant troops from the city, Covenant naval assets began to glass the city, and one member of Noble Team was killed by sniper fire while redeploying.[16][17]
Redeployed, Noble Team and a small force of Orbital Drop Shock Troopers were ordered to destroy the Covenant-occupied Sword Base in a scorched earth operation. Proceeding into the base, the team encountered Doctor Catherine Elizabeth Halsey, who enlisted their help in defending her laboratory amid the ruins of a Forerunner complex from advancing Covenant forces. After successfully holding off the Covenant forces, Halsey ordered SPARTAN-B312 to transport a fragment of the A.I. Cortana to the UNSC Pillar of Autumn, while Jun-A266 escorted her to CASTLE Base elsewhere on Reach.[18] Even as August 30 dawned, however, a second, much larger Covenant fleet was detected exiting Slipstream Space on the edge of the system.
Third Covenant fleet arrivesEdit
- "Gamma Station, this is Fermion RSO. Unconfirmed reports of an in-system Slipspace rupture."—Fermion Remote Sensing Outpost
- "Impossible. Check your source."—Reach Station Gamma
With UNSC ground forces overwhelmed, and the home fleet fighting against the Covenant armadas already present, all hope of holding Reach was lost when a Covenant fleet of three hundred and fourteen vessels came to reinforce the attack. This combined flotilla, the largest ever recorded at that point in the war, would not only crush the UNSC in space and on the ground, but ultimately take up positions to glass the planet itself.
The approaching Covenant fleet was first detected in slipspace by Remote Scanning Outpost Fermion. The station's commander, Chief Petty Officer McRobb, sent an emergency message to FLEETCOM and ordered self-destruct to prevent the station's data from falling into enemy hands.[19] A few minutes later three hundred and fourteen Covenant ships exited slipspace at the outskirts of the Epsilon Eridani System. WINTER CONTINGENCY was declared, and Admiral Roland Freemont issued file/bravo-tango-beta-five, ordering all ships in the system as well as those in the Jericho and Tantalus systems, to rendezvous at Reach in preparation for the coming assault.[20] The Covenant moved in on the orbital defences before the UNSC fleet fully consolidated. Fifty two late arriving UNSC ships, including the UNSC Pillar of Autumn, ran a gauntlet of Covenant warships as they attempted to link up with the main fleet. At the beginning of the battle, only about a hundred UNSC ships were readily available to defend Reach.[21]
Space battleEdit
The main Covenant force moved in on the orbital defences. The initial salvo of Plasma Torpedoes was mostly absorbed by three sacrificial refit and repair stations, allowing the defenders to return fire. The Orbital Defence Platforms and a nuclear minefield combined to take down a full third of the Covenant fleet, destroying about a hundred Covenant ships with the first salvo. The vaporized Titanium-A of the refit stations also served to block incoming plasma torpedoes. However, the next Covenant salvo significantly damaged the UNSC fleet, as the Covenant maneuvered for clear shots around the Titanium dust cloud and moved in for the kill. They destroyed a quarter of the Super MAC platforms, further crippling the UNSC orbital defence capabilities, before retreating out of range. A previously unknown warship type, the Covenant supercruiser, carrying an immensely powerful energy projector revealed itself, destroying the UNSC Minotaur before withdrawing temporarily. The attacking fleet withdrew to regroup after deploying hundreds of dropships to the surface. The UNSC fleet pursued and destroyed a large number of the Covenant dropships, but many still made it through and landed on Reach's northern and southern poles.[22]
As the surviving dropships disgorged thousands of troops, the Covenant fleet began another attack. Some ships made pinpoint slipspace jumps, putting them within the UNSC formation. This left them vulnerable for a short time but allowed them to strike the ODPs directly. The Supercruiser returned and destroyed the UNSC Herodotus and UNSC Musashi from beyond ODP range. The UNSC Pillar of Autumn engaged and destroyed the Supercruiser, detonating a Shiva nuclear device inside the Covenant warship's shields. The detonation reverberated off the shield and disintegrated the Supercruiser.[23][24]
Attack on the orbital defense generatorsEdit
- "There are thousands of them. Grunts, Jackals and their warrior Elites. They have tanks and fliers. Christ, they've breached the perimeter. Fall back! Fall back! If anyone can hear this: the Covenant is ground-side. Massing near the armory!"
Thousands of Covenant landed on Reach in an attempt to destroy the Orbital Defense Generators, and were intercepted by Marine forces who were able to defend against the first few waves of Covenant forces while sustaining heavy casualties. The remaining SPARTAN-IIs who were not assigned to Reach Station Gamma were sent to Orbital Defense Generator Facility A-331 to assist in the defense of the station. Unfortunately, their Pelican escort, Bravo 001, was shot down, resulting in the deaths of four Spartans (including Malcolm-059), and leaving others wounded. Near their crash site, the surviving Spartans discovered the shell-shocked remnants of Charlie Company.[27][24]
Charlie Company had been assigned by Vice Admiral Danforth Whitcomb to find the prototype NOVA Bombs. They were forced to help defend the Orbital Defence Facilities on their way to their objective. Unfortunately, as the Covenant neared the Orbital Defense Generators, someone at HIGHCOM panicked and ordered Longswords to bomb everything within 500 metres, resulting in the destruction of the Covenant ground forces in that location, but also catastrophic friendly fire: Charlie Company was reduced to four men, and its leaders: Lieutenants Jake Chapman and Buckman were killed. The Spartans, after being briefed on the situation by Charlie Company, responded to a distress call from Whitcomb, who requested immediate evacuation. Frederic-104, commander of the Spartans, split the remains of Red Team into four groups: Team Alpha (Frederic-104, Kelly-087, and Joshua-029), which was tasked with eliminating an encampment of 10,000 Covenant and their hovering cruiser (without the use of a nuclear weapon, as the EMP blast would render the generator inoperable), Team Beta, tasked with defense of the Orbital Defense Generators, Team Gamma (Li-008, Anton-044, and Grace-093), ordered to retrieve Whitcomb, and Team Delta (the Charlie Company Marines and six wounded Spartans which included William-043, Isaac-039, and Vinh-030), ordered to secure the Spartans' fallback point at CASTLE Base.[28]
Team Alpha hijacked three Banshees and approached the Covenant encampment (they were ignored by the Grunt Zawaz, who assumed they were Elites on a secret mission) and used Fury Tactical Nuclear Weapons within the shields of the Covenant ship, destroying the encampment and negating the EMP effect that would have disabled the Orbital Defense Generators. Joshua was killed in the process by concentrated small arms fire from the 10,000 Covenant ground forces encamped around the Cruiser.[29] The remaining Spartans of Team Alpha then fell back to CASTLE Base, blasting their way through the remaining Covenant in the area with two commandeered Wraiths. Team Gamma accomplished its mission and fell back to Camp Independence with Whitcomb, where they survived the partial glassing of the planet. Team Delta fell back to CASTLE Base, but in the process lost the remaining Charlie Company marines and every Spartan save for Vinh, Isaac, and William. When the remnants of Team Alpha and Team Delta arrived at CASTLE Base, they found Catherine Halsey there.[30][24]
Unfortunately, Team Beta was unable to stop the Covenant, who attacked in their thousands. The Orbital Defense Generators were compromised, and the Covenant (after eliminating the powerless, immobile ODPs in geosynchronous orbit around the planet) began the glassing of Reach.[31]
Mission to Reach Station GammaEdit
During the space battle, the AI Doppler, controller of Reach Station Gamma, was unable to destroy the vital information on board the UNSC Circumference, an ONI Prowler involved in Operation: HYPODERMIC. As the Covenant deployed troops to the station, Doppler reported this violation of the Cole Protocol to the Pillar of Autumn before self-destructing himself to keep from further violation. In response, Captain Jacob Keyes sent John-117, James-005, and Linda-058 to the station. They accomplished their task and destroyed the NAV database onboard Reach Station Gamma, but at the cost of James and Linda. While on board, they rescued some Marines on board the station: Staff Sergeant Avery Junior Johnson and Privates Wallace Jenkins, Bisenti, and O'Brien. They then were evacuated by Pelican back to the Pillar of Autumn. Linda-058 was clinically dead, but there was a chance of saving her, so she was placed in a cryo-chamber. John-117 asked Lieutenant Hall to scan for James (who was blown into space) but they were unable to find him.[32][24]
Battle at AszodEdit
- Main article: Battle of Aszod
With the battle for Reach now nearing its conclusion, the Pillar of Autumn set down at the Aszod ship breaking yards for repairs as well as to receive the fragment of Cortana that Halsey had tasked Noble Team with delivering to Keyes. Covenant forces soon descended on the shipyards and began landing troops. Although the docked Autumn managed to hold off the Covenant attackers, most of the facility and the surrounding area was occupied.
The remnants of Noble Team, consisting of Carter-A259, Emile-A239, and SPARTAN-B312, arrived on the outskirts of the facility. Carter sent Noble Six and Emile to deliver the package to Keyes. He then sacrificed himself to destroy a Scarab right before it fired its main cannon at Noble Six and Emile. Six and Emile fought their way to the shipyards, where they linked up with some surviving UNSC ground troops. Keyes then contacted the SPARTANs and ordered them to clear Landing Platform Delta so that he could pick up the package. Emile took charge of a mass driver and used it to provide cover fire for the Autumn from the attacking Banshees and Phantoms. Six, meanwhile, cleared the platform of Covenant troops.
After Six was able to clear the platform, Captain Keyes came down in one of two Pelicans, successfully avoiding the dogfight that raged over the Pillar of Autumn and personally took possession of the package from Noble Six. Just then a CCS-class battlecruiser appeared and started heading towards the Autumn. As Emile prepared to fire at the ship, a Phantom, guns blazing, shot down one of the Pelicans and headed towards the mass driver before Emile had time to shoot it down. There, it dropped off the Field Marshall and Sangheili Zealots which were sent to prevent the mass driver from shooting down the battlecruiser. Emile was able to kill one of the Zealots with his shotgun but was mortally wounded when a second one came from behind and stabbed him in the back with an energy sword. With the remaining strength he had, Emile stabbed the Zealot in the neck and fell off the mass driver, taking the Elite with him. With the cruiser closing in on the Autumn, Noble Six volunteered to stay behind to take charge of the gun.
Keyes returned to the Autumn with the package and prepared the ship for take off. Meanwhile, Noble Six was able to kill the last few remaining Zealots and the Field Marshall before heading towards the mass driver. Six took possession of the mass driver and destroyed several Phantoms and Banshees which were directly attacking the mass driver in an attempt to stop Six from shooting at the Cruiser. As the Cruiser came into Six's range and prepared to destroy the Autumn with its energy projector, Six fired directly into the ship's exposed glassing port. The resulting explosion crippled the cruiser, which crash landed on the planet's surface.
With the skies cleared of hostiles, the Pillar of Autumn took off and escaped Reach. Once in space, it made a slipspace jump to Installation 04 (secretly arranged by Cortana).[33] A dozen Covenant ships led by Covenant Supreme Commander Thel 'Vadamee pursued, while the remaining vessels stayed behind to finish glassing Reach. Back at the shipyards, the stranded Noble Six continued fighting against the Covenant invaders. Eventually, the lone SPARTAN was overwhelmed and killed by a group of seven Sangheili warriors.[34]AftermathEdit
- "We are not wasting a single UNSC ship to confirm what we have already seen a dozen times before: Reach is gone. Everything on it is blasted to bits, burned, glassed over, and vaporized. Everyone on Reach is dead. Dead. Dead. Dead."
The UNSC lost their second largest military-industrial complex, after the Sol System, and sustained crippling military losses at Reach, losing over one hundred ships, including irreplaceable cruisers and supercarriers such as the UNSC Basra, UNSC Trafalgar, and the UNSC Musashi, not to mention the large numbers of civilian and military personnel killed by the invading Covenant forces. With the Epsilon Eridani System in Covenant hands, there were no further major stop points before Sol. Some estimates put total defeat at a matter of weeks. The Office of Naval Intelligence believed that the location of Sol was compromised and began relocating heavy industry facilities and command centres out of the system.[36]
At the same time, despite such a devastating defeat the UNSC still fared better than in previous battles. Even against the technologically superior Fleet of Particular Justice, outnumbered 2:1, they still managed to inflict disproportionate losses to the Covenant, due largely to the twenty Orbital Defense Platforms orbiting Reach. The AI Cortana piloted the fleeing UNSC Pillar of Autumn to the previously undiscovered Installation 04.[24] While the Battle of Installation 04 was tangential to the main front, the containment of the initial Flood outbreak-and the experience gained-was just as critical to the survival of the UNSC.
Rescue operationEdit
- Main article: Raid of Reach
As the Covenant began glassing Reach, the surviving Spartans continued their fight. Team Gamma; a Red team splinter composed of Anton-044; Grace-093 and Li-008, had managed to survive and complete their mission to rescue Vice Admiral Danforth Whitcomb from HIGHCOM. Too late to reach CASTLE base, they hid in the mountains attacking Covenant units with the use of guerrilla tactics as opposed to a conventional assault. They programmed their MJOLNIR suits to transmit the "Oly Oly Oxen Free" secret code on E-Band radio, hoping that any surviving SPARTANs would discover it and help them.[37]
Meanwhile underneath the ruins of CASTLE base, Dr. Halsey and 5 SPARTANs (Fred; Kelly; Will; Vinh and Issac) had spent 5 days combing the remains of a Forerunner structure and an additional 5 days trapped in a hallway inside the ruins by Covenant forces. They managed to retrieve a Forerunner Crystal that the Covenant were seeking, but it emitted a burst of radiation which alerted the Covenant to their presence. Vinh and Issac fell at the hands of overwhelming Covenant forces, and the others escaped by blowing the hallway down behind them.[38]
After the survivors were recovered by Ascendant Justice, a Covenant Flagship captured by John-117 after the Battle of Installation 04, the ship drew a group of Covenant vessels out of the system in pursuit to Eridanus Secundus, leading to the destruction of the asteroid colony, and the destruction of the artifact and the recovery of a small number of pieces by the Covenant. Also, the Ascendant Justice destroyed around sixteen Covenant warships during the escape in Slipspace and at the rebel base.[39][40]
July 24, 2552Edit
- 0728 Hours: Noble Team is ordered to investigate a possible Insurrectionist activity at the disabled Visegrád Relay. Shortly after, the team learns that the Covenant are responsible for the attack on the relay. After retaking the relay, Noble Team discovers a data module on one of the dead staff. WINTER CONTINGENCY is declared.[11]
July 26, 2552Edit
- 1128 hours: A SDV-class heavy corvette attacks ONI Sword Base. Noble Team is deployed to secure the base and surrounding outposts. After the corvette is destroyed by orbital bombardment, Noble Team meets up with Dr. Halsey and hands over the data module retrieved from Visegrád Relay.[12]
August 12th, 2552Edit
- 0800 hours: The UNSC Army begins a full scale assault on the Viery Territory. Noble Team is sent to gather intelligence of Covenant-occupied sites and to destroy a EMP-emitting Covenant tower.[13] The UNSC learn too late that the tower served as a teleporter linked to a cloaked Covenant Supercarrier. The UNSC Grafton is destroyed by the Supercarrier.[14]
- 2007 hours: Covenant invasion of Reach is fully underway. Noble Team begins developing a plan to destroy the enemy Supercarrier with a Shaw-Fujikawa Translight Engine used as a makeshift bomb. Auntie Dot informs Noble Team that 60% of the UNSC fleet is in route to Reach, the first battlegroup is set to enter the system in 48 hours.
August 14th, 2552Edit
- 1248 hours: Noble Team assaults and assists UNSC Army troopers in the Sabre Launch Facility and use it as a staging point for a battle above Reach. Using the Slipspace Drive from the UNSC Savannah, Noble Team disables and boards one of the Covenant corvettes and use it as the detonation point for the bomb. Due to a malfunction with the triggering mechanism, a member of Noble Team is forced to detonate the bomb manually. The Supercarrier is destroyed just as a Covenant Fleet arrives above Reach.[15]
- 1248 hours: Covenant forces once again attack Sword Base and occupy the site. Dr. Halsey escapes capture by hiding under the facility.
August 23, 2552Edit
- 1534 hours: The Covenant fleet begins glassing targeted areas of Reach, and a battle breaks out in New Alexandria. Noble Six and remaining Army infantrymen assist in the evacuation of civilians from the city.[16]
- 1857 hours: Noble Six links up with the rest of Noble Team. The team destroys three jammers installed by the Covenant, allowing the UNSC to safely evacuate all personnel from the ONI HQ. When the team reunites, they are debriefed by their supervisor to proceed to Sword Base. Catherine-B320 of Noble Team is killed by a Needle Rifle.[17]
August 26, 2552Edit
- The remnants of Noble Team are extracted by a Pelican from the ruins of New Alexandria.
August 29, 2552Edit
- SPARTAN-B312 and a team of ODSTs secure a landing zone for Noble Team's Falcon. Later Noble Team proceed inside Sword Base only to learn their true mission: escort a package, from Halsey, to the Pillar of Autumn.[18]
August 30, 2552Edit
- 0447 Hours: The Remote Scanning Outpost Fermion detects more incoming Covenant vessels. An emergency message is relayed to FLEETCOM. The outpost then self-destructs per the Cole Protocol due to an unsecured science library and nearby Covenant forces.[41]
- 0500 Hours: Another fleet of 314 Covenant vessels exits slipspace on the edge of the system.[42][43]
- 0519 Hours: Admiral Roland Freemont (FLEETCOM Sector One Commander) issues a general order to all vessels in the Reach, Jericho, and Tantalus systems. All vessels and stations initiate the Cole Protocol and rush to Rally Point ZULU.[44][45]
- 0534 Hours: Covenant vessels make pinpoint slipspace jumps attempting to destroy any incoming or outgoing human vessels before they can regroup. A Covenant carrier targets the UNSC Pillar of Autumn, but is destroyed.[46]
- 0545 Hours: The Covenant vessels begin a frontal assault. As the Covenant and human fleets engage above Reach, it seems to be a stalemate, as Covenant ships take out UNSC ships, while the UNSC ships and MAC cannons take out an equal number. The first salvo by the UNSC vessels destroys a third of the remaining Covenant fleet. A Covenant supercruiser arrives and destroys several UNSC ships while outside human weapons range.[46][24]
- 0558 Hours: Station Gamma Dockmaster AI Doppler is unable to implement the Cole Protocol on the UNSC Circumference, an ONI prowler docked at Reach Station Gamma, due to measures taken to maintain the secrecy of Operation: HYPODERMIC. Doppler sends a priority message to FLEETCOM concerning this violation. Covenant intrusion software detects this oversight, and the Covenant deploys troops to seize the NAV database aboard the Circumference.[47]
- 0616 Hours: The SPARTAN-IIs are dispatched from the Pillar of Autumn. John-117, Linda-058, and James-005 form Blue Team and go aboard the Dock to destroy the Circumference, while the remaining SPARTAN-IIs form Red Team and head down to the surface of Reach to defend the power generators for the MAC guns. James goes MIA when a Needler hits his thruster pack and propels him into space during combat. Blue Team continue through the station, rescuing Staff Sergeant Johnson and Privates O'Brien, Bisenti, and Jenkins from Kig-yar and Sangheili.[48][49][50][24]
- 0620 Hours: Most of the Covenant fleet retreats from the system after deploying more dropships toward the surface. The UNSC fleet fires on the ships, destroying many although hundreds still land on Reach.[51]
- 0621 Hours: The boarding craft are landed and the armory is overrun; the Covenant fleet re-returns to the system. The Covenant split their 150+ remaining ships. One fleet attacks the UNSC ships, while another takes out the MAC cannons and send in troops to land on Reach. The UNSC try to split their 50+ remaining forces to engage the Covenant, but there are too many platforms to cover and too many casualties. The Supercruiser continues to destroy UNSC ships, allowing the other ships to attack the MACs. Eventually the supercruiser was destroyed by the Pillar of Autumn, but the UNSC fleet is practically decimated so it is too late to turn the tide of battle.[52]
- 0631 Hours: Red Team's Pelican is shot down. They have no choice but to jump from the crashing Pelican and make a "hard landing" on the ground. Results in four dead and six seriously injured.[53]
- 0637 Hours: John-117, the clinically dead Linda-058, and the Marines board the Pillar of Autumn.[54][24]
- 0647 Hours: The Pillar of Autumn attempts to retreat from the system[55][24], but remains there for unknown reasons and heads for a drydock at the Aszod ship breaking yards on Reach, possibly.
- 0649 Hours: Red Team sets up defenses around ODG Facility A-331.[56][24]
- 0711 Hours: Fred-104, Joshua-029, and Kelly-087 plant a nuclear warhead through the gravity lift in a landing Covenant cruiser, leaving Joshua-029 MIA, (presumed KIA) in the process.[57]
- 0720 Hours: The Covenant destroys ODG Facility A-331 by aerial bombardment, though some Spartans escape. The surviving MAC guns are now offline and are quickly destroyed by plasma torpedoes. Remaining UNSC vessels have no choice but to retreat. The Covenant warships are split into two groups: five ship teams that hunt down the surviving UNSC vessels and a group of ships that align themselves in formation and begin to glass Reach.
- 1652 hours: The remainder of Noble Team head to the Aszod ship breaking yards to deliver a fragment of Cortana, which holds important navigation data, to the Pillar of Autumn. SPARTAN-B312 successfully delivers the package to the Autumn and allows the vessel to escape by destroying an incoming Covenant battlecruiser. The Pillar of Autumn, pursued by a dozen Covenant vessels, retreats from the system, heading for Installation 04.[33]
- 2000 Hours: SPARTAN-B312 is one of the few standing survivors at the ship-breaking yards of Aszod and the planet Reach. The SPARTAN makes a last stand, fighting off waves of Covenant forces, until eventually being overwhelmed and then finally killed by a squad of 7 Sangheili warriors.[34]
September 2552Edit
- September 7
- 0002 Hours: The SPARTAN Red team survivors and Dr. Halsey, after days of walking through the abandoned Titanium mines below CASTLE base, reach a corridor covered in Forerunner symbols.[24]
- September 11–12
- Unknown time: The Installation 04 survivors arrive at Reach, having experienced a temporal anomaly, causing them to be sent nearly two weeks into the past, after traversing through Slipspace with a Forerunner Crystal.[58]
CovenantEditSpace AssetsEditCarriers
Corvettes Cruisers Fleets TriviaEdit
GalleryEditList of appearancesEdit


text:
Hello, we are upgrading AutoCAD Map 3D from 2009 to 2012 and I am wondering what is the correct workflow. We have the network license version. Do I need to update the network license manager in some way before I install the software on workstations, or does it work with the old licensemanager??
The setup of the server should be the same. I would update the server license tools to 11.9.
The link below for the IPv6 are *.exe files, I would create backup the old IPv4 tools after installing and replace them:
Log into access your profile, ask and answer questions, share ideas and more. Haven't signed up yet? Register
Start with some of our most frequented solutions to get help installing your software.
CAD Managers |
Installation & Licensing |


text:
- 10-02-2013, 08:06 AM Thread Author #1
Galaxy Note 3 - Anxiously waiting!
For tmobile to open. 1 hour to go. Called last night to confirm they will have the phones for sale, was told yes. Will call before I leave to reconfirm and hopefully they can put one on reserve. It's not an iphone so I'm not to worried about long lines.
Re: Galaxy Note 3 - Anxiously waiting!
Hopefully you were able to pick it up, I'm assuming from not another post that you did! I know that I went to my local tmo and they had 24 left after me, after getting in 25. There wasn't a soul in the store. They also said that white is on back order for at least a month or so. But back to setting up the beauty!
Similar Threads
HELP: Note 3 Developer Options Bug - "Transition Animation Scale"
By sirbljm in forum Samsung Galaxy Note 3Replies: 40Last Post: 02-21-2014, 11:04 AM -
Galaxy Note 3 - Note 2 to Note 3 Transfer
By mayfieldh in forum Samsung Galaxy Note 3Replies: 12Last Post: 11-29-2013, 09:49 AM -
HELP: Note 3 Developer Options Bug - "Transition Animation Scale"
By sirbljm in forum Android 4.1 / 4.2 / 4.3 Jelly BeanReplies: 3Last Post: 10-29-2013, 09:22 AM -
could the note 3 be the phone im looking for?
By RavenSword in forum General News & DiscussionReplies: 4Last Post: 10-03-2013, 12:43 PM -
Galaxy S4 Charger/Cable Not Working - Help!
By MrGalaxyS4 in forum Samsung Galaxy S4Replies: 2Last Post: 10-02-2013, 01:27 PM