telomeres are the dna  protein complexes at the ends of linear chromosomes that solve the problem of replicating chromosome ends by semiconservative dna replication . they also protect the native chromosome ends from the dna repair pathways that act on ends generated by double - strand breaks . evolution has solved this issue differently in prokaryotes and in eukaryotes . in several bacteria and viruses , replication produces inverted repeats that are processed into 2 covalently closed hairpins by a resolvase . in eukaryotes , telomeres are stable open double - strand ends and are therefore at risk of being erroneous substrates for dna double - strand break repair . two pathways efficiently repair double - strand breaks : nonhomologous end joining ( nhej ) and homologous recombination . homologous recombination is a more complex process that uses a template sequence for repair , most often the sister chromatid in mitotic cells . since telomeric dna are tandem arrays of short duplex repeats in the same orientation relative to the chromosome ends , homologous recombination events between telomeres can elongate or shorten telomeres , but can not fuse them . in other words , a recombination event at telomeres is without immediate consequence on chromosome structure and remains reversible as long as the telomere length stays above a minimum threshold . homologous recombination at telomeres is not fully repressed in normal cells and may play a physiological role in telomere length homeostasis , in particular when telomere length has shifted far from equilibrium or its usual range . in contrast , a single end - joining event mediated by nhej between 2 telomeres fuses them , generating either a dicentric chromosome or a circular chromosome , the latter being at risk of being converted into a dicentric through sister chromatid exchange . dicentrics are unstable and can result in deleterious and potentially oncogenic copy number aberrations through several mechanisms such as breakage - fusion - bridge cycles , chromothrypsis , and polyploidization . thus , the existence of a stable karyotype of linear chromosomes in all cells where the nhej pathway is active requires that nhej is fully prevented from acting on telomeres . it should be noted that chromosome end fusions can occur with or without telomeric sequences at the fusion . in the first case , telomere function failed despite the native sequence . how such telomere fusions with native telomere sequences are prevented is the topic of this review . in contrast , when all or most of the telomere repeats are missing , the fusion is a secondary consequence of telomere loss . in such cases , prevention of fusion relies on the mechanisms ensuring proper telomere sequence maintenance and replication , which will not be covered here . the first is often called c - nhej ( classic or canonical nhej ) and is an efficient pathway in which a dedicated dna ligase , lig4 , seals the double - strand ends . here , very limited base pairing between single - strand 3 tails allows ligation by lig3 ( the base excision repair ligase , absent in yeast ) or eventual priming of dna synthesis , the subsequent ligation being carried out by lig1 ( the main replicative ligase , ubiquitous in eukaryotes ) but not lig4 . a - nhej is inhibited by replication protein a , and is mostly seen in circumstances where c - nhej and homologous recombination can not act ( e.g. , in mutant contexts ) . a - nhej might be a back up for these normally efficient pathways or the accidental by - product of various activities acting on dna in the nucleus , a sort of biochemical noise without a positive function in normal cells . in this review , for example , it is transiently shut down during mitosis in mammals   and during s / g2 in the fission yeast schizosaccharomyces pombe . in the budding yeast saccharomyces cerevisiae , nhej is an haploid - specific pathway and is repressed in diploid cells . in the distantly related yeast lachancea kluyveri , nhej was lost during evolution . the problem arising from the coexistence of telomeres and nhej is thus relieved in these contexts . the function of end joining is to reseal dna ends formed by double - strand breaks . its substrates are double - stranded ends that can be either blunt or possess short single - strand overhangs of a few nucleotides . dna ends with short and perfectly cohesive overhangs can be repaired very efficiently and accurately by nhej . if nucleotides at the ends are damaged ( e.g. , at breaks generated by ionizing radiation ) , end joining can still process and repair the broken ends but the native sequences may not be restored . telomeres are composed of an oriented g - rich repeated motif ( e.g. , tg13 in s. cerevisiae and ttaggg in vertebrates ) . they are mostly double - stranded , but display a short terminal 3 single - strand overhang of approximately 510  nt in budding yeast and 20300  nt in vertebrates . thus , 2 telomeres will not form short cohesive overhangs . if anything else , their fusion by nhej would be a relatively slow act involving extensive processing of the ends . this kinetic effect might be the first barrier against fusions with which other mechanisms synergize . if the ends remain unrepaired , several nuclease activities degrade the 5 ends to generate single - strand 3 tails that are no longer substrates for nhej but are instead committed to homologous recombination . thus , it should be protective to maintain stable 3 single - strand overhangs at telomeres that are long enough not to be a substrate for nhej . this maintenance results from a regulated equilibrium between 5 degradation and dna synthesis priming on the single - strand g - rich tails . in mammals , stability of the 5 strand at the broken ends requires the recruitment of 53bp1 through a series of local histone modifications by atm , rnf8 , and rnf168 . 53bp1 protects 5 ends through at least 2 effectors  rif1 and ptip  that either prevent 5 degradation or reverse it through dna synthesis . 53bp1 is important for nhej events requiring relatively complex processing of the ends , in particular the fusions of ends from distinct breaks . the relative slowness of these reactions may explain why they strongly depend on the extensive 5 stability established by 53bp1 . telomere fusions also require 53bp1 . in normal cells , 53bp1 is excluded from telomeres . 53bp1 also helps to keep ends mobile . whether this is a consequence of increased mobility of double - stranded ends versus single - stranded ends is unknown , but since distant ends must meet in order to be fused by nhej movement within the nucleus can be a key limiting step in telomere fusions . through the exclusion of 53bp1 in addition to a simple 3 single - strand overhang , the telomere dna end may adopt unusual conformations to escape nhej . in one such case , the 3 single - strand telomere end loops back to hybridize with the complementary strand of double - stranded telomere repeats , forming an intramolecular d - loop called the t - loop . this strand invasion reaction further engages the telomere end into a reversible homologous recombination intermediate . this shifts the issue from preventing nhej to preventing further commitment to homologous recombination ( that is 3 extension ) and keeping this structure stable ( a d - loop is normally transient and unfolded by helicases or cut by nucleases ) . a likely gain from this structure is that it is less problematic to proceed into a full homologous recombination event once in a while ( causing only telomere elongation or shortening ) than to generate a single telomere fusion . in other words , it seems best to chose the less harmful of 2 bad events . the 3 telomere single - strand tails are also sufficiently g rich to form g - quadruplexes that could oppose nhej . the t - loop and g - quadruplex structures may also help to protect the 3 telomere single - strand tails from a - nhej . in yeast , the 3 telomere single - strand tails are probably too short to form stable t - loops or g - quadruplexes structures , but they may also be too short for a - nhej . similarly , in arabidopsis a large fraction of telomeres are blunt - ended and unlikely to adopt a secondary structure . in some species , including s. cerevisiae and ciliates , a - nhej is also prevented by the lack of base pairing between 3 telomere overhangs . an abundant protein , it binds very rapidly to the broken ends of a double - strand break . whether more than one ku molecule binds to each end is unclear . at the minimum , the function of ku is to restrain 5 resection and control the recruitment of mre11 and lig4 . it probably also provides part of the scaffold that might align the ends and bring them in close proximity with the correct phasing to facilitate ligation by lig4 . unfortunately , this is still speculative and we do not have a complete understanding of the roles of ku during the nhej process . this complex binds to double - stranded dna ends and might help to tether the ends together as already mentioned , lig4 is an atp - dependent dna ligase dedicated to nhej . the 2 lig4 co - factors are lif1 and nej1 in budding yeast , and xrcc4 and xlf / cernu in mammals . xrcc4-xlf can form polymers in vitro and may thus act as a splint bridging the 2 broken ends , a model that remains to be tested in vivo . in addition to these core factors , nhej can mobilize a dna polymerase from the pol - x family to fill in short gaps at the ends prior to ligation : pol4 in budding yeast ; pol  and pol  in mammals . in budding yeast , as at double - strand breaks , one function of ku at telomeres is to limit 5 resection , thus helping to set the size of the 3 single - strand telomere overhang . in budding yeast , ku also helps to recruit telomerase and a heterochromatin factor , sir4 , at telomeres . in contrast to ku , the mre11 complex , lig4 , and its co - factors are absent from telomeres . this indicates that nhej inhibition is in part based on their exclusion from telomeres , or at least their inability to form a stable complex with telomere dna ends . an exception is during replication , when mre11 can be transiently recruited to telomeres . at this time , mre11 is important for telomere elongation by telomerase , at least in budding yeast where the bulk of elongation depends upon mre11 and its associated checkpoint kinase tel1 . in particular , mre11 plays an important role in replication - coupled 5 resection at telomeres , a key step in generating the 3 single - stranded tails that telomerase can elongate . thus , during telomere replication the inhibition of nhej must rely on mechanisms other than mre11 exclusion . the telomere repeated motifs allow the concentration of specialized proteins that recognize them and establish telomeric functions . perhaps not surprisingly , proteins bound to the double - stranded telomeric repeats and the factors recruited by these proteins are essential to protect telomeres from nhej in all species in which this question has been addressed . in the fission yeast s. pombe , the taz1 protein binds telomeric dna and recruits the rap1 protein . in the absence of taz1 or rap1 , telomeres fuse together in a process that requires lig4 , ku , and mre11 . thus , both taz1 and rap1 are required for nhej inhibition , but how these proteins function is unknown . in mammals , 2 taz1 orthologs , trf1 and trf2 , bind double - stranded telomeric dna . telomeres fuse extensively in a ku , lig4 , and mre11-dependent manner indicating that trf2 is essential for nhej inhibition . in cells lacking rap1 , telomere fusions remain below detection threshold . however , in cells where trf2 is detached from telomeres , artificial tethering of rap1 to the telomeres re - establishes nhej inhibition independently of trf2 . this suggests that trf2 protects telomeres from nhej through multiple mechanisms , at least one of which involves rap1 and the others being rap1-independent . trf1 also contributes to nhej inhibition , perhaps indirectly by favoring trf2 assembly or function . first trf2 , but not rap1 , is essential to exclude 53bp1 from telomeres through the synergistic inhibition of atm and rnf168 . inhibition of nhej by trf2 in complex with rap1 can be established in vitro on short double - stranded substrates , suggesting that the rap1-dependent pathway is not related to a structure established by the 3 telomere single - strand overhang . trf2 also directly interacts with ku and this interaction might help to inhibit nhej by preventing a ku self - association that is predicted to bridge dna ends during the nhej process . in addition , trf2 protects telomeric 3 single - strand overhangs from degradation by the nuclease activities of the xpf / ercc1 and mre11 complexes . trf2 also generates t - loops independently of rap1 in vitro and in vivo , in part through promotion of supercoiling and strand invasion and in part through protection of the holliday junctions . it should be noted that t - loops were observed after psoralen crosslinking in isolated nuclei so it remains formally possible that their accumulation is a trf2-dependent artifact generated during nuclei isolation and that such structures are transient and unfolded by helicases in living cells . in s. cerevisiae , there is no ortholog of taz1/trf1/trf2 and the rap1 protein directly binds the telomere sequences at a density of approximately 1520 molcules per telomere . in yeast cells lacking rap1 , the telomeres fuse in a ku , lig4 , and mre11-dependent manner . one requires the rap1-interacting factor rif2 , an aaa+ protein originating from a recent duplication of an orc subunit gene in the yeast lineage . first , rif2 inhibits nhej at a double - strand break in the absence of telomeric dna when it is artificially targeted there by a domain of rap1 . this suggests that rif2 acts on protein complexes and not through a telomere - specific dna structure . it also shows that nhej inhibition by rif2 is a standard cis effect in which the recruitment of a factor locally inhibits a molecular function . second , rif2 inhibits not only nhej at telomeres , but also all other mre11-dependent processes ( 5 resection , telomerase - mediated telomere elongation and a pathway of homologous recombination between telomeres ) . the simplest hypothesis is that rif2 inhibits the mre11 complex ; this single molecular activity would explain the multifunctionality of rif2 . the origin of rif2 suggests that orc may play a similar role at telomeres in other eukaryotes , for example in human cells where orc is recruited to telomeres by trf2 . independently of rif2 , in budding yeast rap1 inhibits nhej at telomeres through recruitment of the heterochromatin factor sir4 , a yeast - specific protein of unknown evolutionary origin . interestingly , sir4 interacts directly with ku . this interaction is essential for stable sir4 recruitment at telomeres but may also inhibit nhej , for example by disrupting the assembly of lig4 on ku molecules bound to dna ends . although this scenario remains speculative , the parallel with the trf2-ku interaction in mammals is striking . rap1 in budding yeast also inhibits nhej independently of rif2 and sir4 through a region of the protein that includes its dna binding domain . dna binding by rap1 may directly out - compete the stable assembly of mre11 on dna ends , favor a dna secondary structure that is resistant to nhej and 5 degradation , or recruit unidentified effectors . we recently found that the yeast translocase and sumo - dependent ubiquitin ligase uls1 are essential to maintain nhej inhibition at telomeres . uls1 does not act in a specific inhibitory pathway ; instead , its role is to ensure that rap1 function is maintained . specifically , uls1 eliminates rare non - functional poly - sumoylated rap1 molecules bound to telomeres . how poly - sumoylation cripples the ability of rap1 to inhibit nhej is unknown . thus , in both mammals and in yeast , multiple mechanisms cooperate to fully inhibit nhej at telomeres . their synergy is reinforced by the multiplicity of dna - bound molecules at each telomere and ensures that nhej inhibition at telomeres is continuously efficient and resilient to normal telomere length fluctuation . in budding yeast , the protection against telomere fusions goes one step further : dicentrics formed by telomere fusions often break at the fusions during mitosis , a process that restores the parental chromosomes . by allowing reversibility of telomere fusions , this rescue pathway can back up a temporary lapse of nhej inhibition at telomeres , further protecting the cell from the deleterious consequences of an unstable karyotype . the author would like to thank karine dubrana , madalena tarsounas and pablo radicella for helpful criticism and advice on this review . studies in the author 's laboratory are supported by grants from arc and anr ( blanc - svse-8 - 2011-telo&dicens ) .
the telomeres of eukaryotes are stable open double - strand ends that coexist with nonhomologous end joining ( nhej ) , the repair pathway that directly ligates dna ends generated by double - strand breaks . since a single end - joining event between 2 telomeres generates a circular chromosome or an unstable dicentric chromosome , nhej must be prevented from acting on telomeres . 
 multiple mechanisms mediated by telomere factors act in synergy to achieve this inhibition .