the initiation of dna replication is tightly regulated so that origins of replication are normally utilized only once per cell cycle . many of the factors and regulators that participate in the initiation phase of dna replication are known , and the order in which these factors interact with one another and load onto origins is emerging . the process begins in late mitosis or g1 , when origin recognition complex ( orc ) * binds directly to chromosomal origins of replication ( for review see kelly and brown , 2000 ; bell and dutta , 2002 ) . once bound , orc recruits cdc6 and cdt1 , which in turn allow assembly of the mini - chromosome maintenance ( mcm)2 - 7 complex onto the dna . recruitment of mcm2 - 7 , the presumptive replicative helicase ( labib and diffley , 2001 ) , completes assembly of the prereplication complex ( pre - rc ) . at the g1/s transition , the pre - rc is converted to a preinitiation complex ( pre - ic ) through the actions of two protein kinases , s phase specific cyclin dependent kinase ( s - cdk ) and the cdc7-dbf4 protein kinase , which cooperate to recruit the initiation factor cdc45 ( mimura and takisawa , 1998 ; zou and stillman , 1998 , 2000 ; jares and blow , 2000 ; walter , 2000 ) . in addition to s - cdk and cdc7 , another factor , mcm10 , has been shown in xenopus extracts to act after mcm2 - 7 to recruit cdc45 ( wohlschlegel et al . , 2002 ) . after cdc45 binding , the next discernible step in the initiation process is origin unwinding and recruitment of the single - stranded dna binding protein rpa , followed by dna polymerase  ( pol  ; mimura and takisawa , 1998 ; tanaka and nasmyth , 1998 ; zou and stillman , 1998 , 2000 ; mimura et al . , 2000 ; walter and newport , 2000 ) . because cdc45 loading is the last known step before origin unwinding and the commencement of dna synthesis , and because it has been shown to be rate limiting for dna replication in xenopus egg extracts ( edwards et al . , 2002 ) , this event is considered to be critical for the regulation of initiation . the drosophila mus101 gene was isolated 25 years ago in a screen for mutants showing hypersensitivity to dna damaging agents ( boyd et al . , 1976 ) . hypomorphic alleles have been isolated that exhibit defects in the kinetics of progression through s phase , and in chorion gene amplification ( orr et al . , 1984 ) . therefore , these two phenotypes are consistent with a role for mus101 in dna replication . molecular cloning of mus101 ( yamamoto et al . , 2000 ) showed that the gene encodes a protein composed of reiterated brca1 cooh terminus ( brct ) domains , and that it is highly related to a human protein named topbp1 , a putative dna damage response protein ( yamane et al . mus101/topbp1 is also distantly related to two yeast genes known to be required for dna replication , budding yeast dpb11 ( araki et al . , 1995 ) and fission yeast cut5 ( saka and yanagida , 1993 ) . in dna replication , dpb11 is thought to act after cdc45 loading , and rpa binding , to recruit pol  and pol  to the unwound origin ( masumoto et al . , 2000 ) . this role for dpb11 is supported by the findings that dpb11 requires mcm2 - 7 and rpa to associate with the origin , that dpb11 is required for origin binding of pol  and pol  , but not rpa , and that dpb11 interacts both genetically and physically with pol epsilon ( araki et al . in addition to their dna replication functions , both dpb11 ( araki et al . , 1995 ; wang and elledge , 1999 ) and cut5 ( saka and yanagida , 1993 ; saka et al . , it is not known if mus101/topbp1 shares with dpb11/cut5 a role in checkpoint control . in order to uncover the mus101 function in dna replication , we have isolated the xenopus mus101 protein and used xenopus egg extracts to identify the mus101-dependent step in dna replication . the results yield the surprising conclusion that mus101 functions to load cdc45 onto replication origins , and that it does so in a manner distinct from the other known cdc45 loading factors . the metazoan mus101 protein family includes human topbp1 , drosophila mus101 , and an uncharacterized locus in c. elegans , f37d6.1 ( fig . 1 
 
   a ) . to initiate an analysis of this protein family in dna replication , we isolated a xenopus homologue of mus101 , named xmus101 . the full - length xmus101 cdna encodes a 1,513 amino acid protein with significant similarity to topbp1 ( 75% amino acid similarity ) , to drosophila mus101 ( 43% similarity ) , and to c. elegans f37d6.1 ( 39% similarity ) . the similarity between xmus101 and cut5 is restricted to the brct domains , whereas no significant sequence similarity between xmus101 and dpb11 was detected . ( a ) the mus101 protein family . shown are schematic depictions of mus101-related proteins from divergent organisms . the shaded gray boxes indicate the position of the brct domains , and the numbers to the right indicate the size of the protein , in amino acids . for comparison , the domain structure of budding yeast dpb11 and fission yeast cut5 is also shown . ( b ) immunoblot analysis of xenopus egg extract ( xee ) , or egg extract that had been immunodepleted of xmus101 ( -xmus101 ) , probed with affinity - purified anti - xmus101 antibodies . the asterisk denotes a background band recognized by the antibody that is not diminished in the depleted extract . the numbers to the left of the gel denote the migration position and molecular mass , in kd , of molecular mass markers . ( c ) egg extracts were prepared , and then depleted of xmus101 protein ( -xmus101 ) or mock - depleted ( mock ) . additionally , xmus101-depleted extracts were supplemented with in vitro transcription and translation ( ivt ) reactions programmed by the xmus101 cdna . in vitro transcription sperm chromatin was then added , along with p - datp , and dna replication in the given extract was measured at the indicated times . the graph depicts the amount of dna replication observed after quantification of the dried gels by phosphoimager analysis . the data shown are from a single experiment , and are representative of four independent trials . the amount of replication observed in the mock - depleted sample at the 90-min time point was set to 100 , and all other values adjusted accordingly . rabbit polyclonal antibodies were raised against a recombinant protein containing the nh2-terminal 333 amino acids of xmus101 , and affinity purified . 1 b ) , and can immunoprecipitate the 180 kd in vitro transcription and translation product of the cloned xmus101 cdna ( unpublished data ) . to determine if xmus101 functions in dna replication in xenopus , the protein was removed from egg extracts , by immunodepletion , and the ability of the depleted extract to replicate sperm chromatin was assessed . as shown in fig . 1 b , the immunodepletion procedure removed all detectable xmus101 from the extract . to control for nonspecific effects of immunodepletion , a mock - depleted extract was also prepared , and replication was measured in both the mock- and xmus101-depleted extracts . dna replication was severely compromised in the xmus101-depleted extract , relative to the mock - depleted control extract ( fig . importantly , addition of recombinant xmus101 protein , produced by transcription and translation in vitro , fully rescued the replication defect in the mus101-depleted extract ( fig . rescue was not observed when transcription was performed in the opposite orientation , to produce the anti - sense xmus101 mrna ( fig this rescue by the sense , but not anti - sense xmus101 mrna , demonstrates that the xmus101-depleted extracts failed to replicate their dna because xmus101 was removed , and not because of nonspecific inactivation of the extract . chromatin immuoprecipitation - based methodologies in budding yeast indicate that dpb11 acts after rpa has bound to the origin to recruit pol  and pol  to the assembling replication complex ( masumoto et al . , 2000 ) . because xmus101 is distantly related to dpb11 , we asked if xmus101 was required for replication complex assembly in egg extracts . for this , sperm chromatin was added to either mock- or xmus101-depleted extract , and after 45 min the chromatin was isolated from the extract and probed by immunoblotting for the presence of components of the replication complex . this experiment shows that loading of the mcm2 - 7 complex onto chromatin was not affected by the absence of xmus101 , as equal amounts of chromatin - associated mcm7 protein were found in the mock - depleted sample relative to the xmus101-depleted sample ( fig . 2 
 
   a , panel viii , lanes 3 and 4 ) . in contrast , there was a dramatic decrease in the amount of pol  , rpa , pol  , and , interestingly , cdc45 associated with chromatin in the xmus101-depleted sample , compared with the mock - depleted sample ( fig . 2 a , panels ii  v , lanes 3 and 4 , respectively ) . this decrease in pol  , rpa , pol  , and cdc45 levels was not observed when total extract was blotted ( fig . 2 a , panels ii v , lanes 1 and 2 , respectively ) , indicating that the decrease was not due to quantitative coprecipitation with xmus101 . we also assessed the ability of cdc7 and mcm10 to bind to chromatin in xmus101-depleted extract , and found that both of these factors were able to assemble into the initiation complex in the absence of xmus101 ( fig . 2 a , panels vi and vii , lanes 3 and 4 ) . based on the data in fig . 2 a , we conclude that xmus101 is required for chromatin - binding of cdc45 , rpa , pol  , and pol  , but not for mcm2 - 7 , cdc7 , or mcm10 . thus , unlike budding yeast dpb11 , xmus101 is a cdc45 loading factor that acts prior to origin unwinding during initiation . xmus101 is required for the loading of cdc45 onto chromatin during the initiation of dna replication . ( a ) immunoblot of either total extract samples from mock - depleted ( mock , lane 1 ) , or xmus101-depleted ( -xmus101 , lane 2 ) extract , or chromatin isolated after a 45-min incubation in either mock- ( mock , lane 3 ) , or xmus101-depleted ( -xmus101 , lane 4 ) extract . the blots were probed with antibodies against xenopus xmus101 ( panel i ) , pol  ( pol  p60 subunit , panel ii ) , rpa ( the 34-kd subunit , panel iii ) , pol  ( pol  p70 subunit , panel iv ) , cdc45 ( panel v ) , mcm10 ( panel vi ) , cdc7 ( panel vii ) , or the mcm7 component of the mcm2 - 7 complex ( panel viii ) . ( b ) immunoblot of complete egg extract , both total extract ( tot . ) and the isolated chromatin fraction ( chr . ) , and membrane - free egg extract , or egg cytosol , both total extract and the isolated chromatin fraction . ( c ) egg extracts were prepared and supplemented with sperm chromatin ( lane 2 ) , or sperm chromatin plus recombinant geminin ( 500 nm , lane 3 ) , or sperm chromatin plus recombinant p27kip ( 500 nm , lane 4 ) . after a 45-min incubation , the chromatin was isolated and probed , by immunoblotting , for the presence of cdc45 and xmus101 . lane 1 shows a reaction lacking sperm chromatin . to better understand how xmus101 participates in cdc45 recruitment we found that xmus101 did not load onto chromatin in membrane - free egg extracts ( egg cytosol ) that are competent to form pre - rcs , but incompetent to form nuclei or execute subsequent initiation events ( fig . in contrast , in nuclear assembly extracts that undergo initiation of replication , xmus101 bound to chromatin ( fig . 2 b , lane 2 ) . the inability to bind to chromatin in egg cytosol indicates that xmus101 is not a constituent of the pre - rc . to ask if xmus101 chromatin binding is dependent upon an intact pre - rc once nuclear formation occurs , we assessed xmus101 chromatin - association in extracts treated with geminin , an inhibitor of the cdt1 protein ( wohlschlegel et al . 2 c , lane 3 ) . to ensure that the geminin was active in this experiment , we also probed the purified chromatin fraction for the presence of cdc45 , and found , as expected ( jares and blow , 2000 ) , that geminin blocked cdc45 association with chromatin ( fig . next , we determined if xmus101 chromatin association required s - cdk activity . for this we used p27kip ( toyoshima and hunter , 1994 ) , an s - cdk inhibitor that has previously been shown to block dna replication in frog egg extracts ( michael et al . , 2000 ) . we found that p27kip prevented cdc45 from loading onto chromatin , as expected , but that it could not prevent loading of xmus101 ( fig . 2 ( b and c ) demonstrate that xmus101 requires nuclear formation to bind to chromatin , but that it binds in a s - cdk and pre - rc  independent manner . therefore , there was a possibility that xmus101 bound to chromatin independently of replication initiation . to test this we have found that removal of orc from conventional nuclear assembly extracts by immunodepletion is difficult . therefore , we turned to a nucleus - free replication system derived from xenopus egg extracts ( walter et al . , 1998 ) . in this system , sperm chromatin is first mixed with membrane - free egg cytosol to stimulate pre - rc assembly . subsequently , a highly concentrated nucleoplasmic extract ( npe ) is added to promote pre - ic assembly and drive the initiation of dna replication . in this way , pre - rc assembly can be experimentally separated from pre - ic assembly and initiation . as reported previously ( walter et al . , 1998 ; wohlschlegel et al . , 2002 ) , when sperm chromatin was incubated in egg cytosol alone , the pre - rc components orc2 and mcm7 bound to the chromatin , but mcm10 did not ( fig . like mcm10 , xmus101 did not bind to sperm chromatin in egg cytosol ( fig . upon addition of npe , both mcm10 and xmus101 were recruited to the chromatin ( fig . 3 a , lane 6 ) . to determine whether xmus101 binding to chromatin required orc , we used orc2 antibodies to deplete the orc complex from egg cytosol and npe . under these conditions , 3 a , lane 8) . as seen in the nuclear assembly system , xmus101 binding in the nucleus - free system was largely independent of chromatin binding by mcm2 - 7 ( fig . we conclude that xmus101 is recruited to replication origins in an orc - dependent manner , which links xmus101 chromatin association to the initiation of dna replication . xmus101 requires orc for association with sperm chromatin . in lanes 14 , egg cytosol ( ec ) that either contained or lacked orc , as indicated , was mixed with sperm chromatin and incubated for 30 min . the chromatin was then isolated and probed by immunoblotting for the presence of xmus101 , mcm10 , mcm7 , or orc2 . in lanes 58 , egg cytosol that either contained or lacked orc , as indicated , was mixed with sperm chromatin and incubated for 30 min . after incubation , npe that either contained or lacked orc , as indicated , was then added and incubation continued for an additional 30 min before isolation of the chromatin and immunoblotting for the indicated factor . the npe contained the replication inhibitor aphidicolin , to trap assembled replication complexes and prevent disassembly following the completion of dna replication . having established that xmus101 is essential for initiation , we next asked if the protein is required during the elongation phase of replication . for this , conventional egg extracts were prepared and incubated with sperm chromatin for 30 min to allow replication complex assembly and initiation to occur . after the 30-min incubation , the chromatin was isolated using isolation buffer containing 100 mm kcl , which removes residual xmus101 protein from the isolated chromatin ( unpublished data ) . the salt extracted chromatin was then transferred to either a mock- or xmus101-depleted extract , and incubation was continued in the presence of radio - labeled datp to measure dna replication ( experimental scheme shown in fig . 4 
 
   a ) . when dna replication was measured in this experiment , no significant difference between the mock- and xmus101-depleted extract was observed ( fig . 4 b ) . it was important to confirm that the xmus101-depleted extract was devoid of xmus101 protein , and that no contaminating xmus101 was transferred along with the sperm chromatin . for this , we probed samples of the mock- and xmus101-depleted extracts after transfer of the salt - extracted chromatin to the given extract , and found no detectable xmus101 in the xmus101-depleted sample ( fig . thus , despite the lack of detectable xmus101 in the xmus101-depleted extract containing the salt - extracted chromatin , dna replication was not affected . 1 and 4 show that when xmus101 is removed before initiation , dna replication fails to occur ; however , when it is removed after the initiation phase has passed , there is little effect on dna replication . these data indicate that xmus101 is an initiation factor , and that it does not function during elongation . ( b ) dna replication was measured in the given extract at the given time . ( c ) immunoblotting of either mock - depleted ( mock ) , or xmus101-depleted ( -xmus101 ) extract after transfer of the chromatin , showing that no detectable xmus101 is transferred along with the chromatin . in this report , we have shown that mus101 is required for the initiation of dna replication , and that it is likely dispensable for the elongation phase of replication . we have also pinpointed the mus101-dependent step in initiation , by showing that cdc45 fails to load onto chromatin in xmus101-depleted frog egg extracts . thus , xmus101 exerts at least one of its functions in the initiation of dna replication upstream of the cdc45 loading step . consistent with the early execution point of xmus101 , we find that it loads onto chromatin , in an orc - dependent manner , at the equivalent of the g1/s boundary in our extracts . that is , its loading does not occur in cytosolic extracts which support pre - rc formation , but only after chromatin has been exposed to an initiation - competent nuclear environment . these data imply that , besides orc , xmus101 chromatin association also requires an unknown condition that is only met inside the nucleus . this could result from loss of a cytosolic inhibitor of xmus101 chromatin association , or be due to a positive influence of the nucleus , such as an unknown bridging factor , or a nucleus - specific alteration of xmus101 activity . alternatively , because the concentration of dna replication factors is much higher within nuclei ( or in npe ) , relative to cytosol , the nuclear - dependence of xmus101 chromatin association might simply result from a concentration effect . consistent with this , we find that the concentration of xmus101 in npe is at least 20-fold higher than in egg cytosol ( unpublished data ) . although origin binding of xmus101 requires orc , it is independent of mcm2 - 7 and s - cdk . this mcm2 - 7 independence distinguishes mus101 from the other known cdc45 loading factors , cdc7 and mcm10 , both of which require a fully intact pre - rc for chromatin loading ( jares and blow , 2000 ; walter , 2000 ; wohlschlegel et al . , 2002 ) . furthermore , we have shown that mcm2 - 7 , cdc7 , and mcm10 all load onto chromatin independent of xmus101 . thus , the mutual independence of xmus101 and mcm2 - 7/cdc7/mcm10 origin association strongly suggests that the assembly pathway that ultimately culminates in origin loading of cdc45 bifurcates at a step soon after chromatin - loading of orc ( fig . 5 ) 
 
  . one pathway leads via mcm2 - 7 to the recruitment of cdc7 and mcm10 , and the other pathway works through xmus101 . besides mcm2 - 7 , mcm10 , cdc7 , and mus101 , the other major requirement for cdc45 loading is s - cdk activity . the model in fig . 5 depicts two pathways emanating from orc , and it is not yet clear which pathway requires s - cdk . we have found that xmus101 loads onto chromatin in the absence of s - cdk activity , suggesting that if s - cdk acts in the mus101 branch , then it would be downstream of mus101 . if s - cdk does act in the mus101 branch of the initiation pathway , then both branches would be under the control of protein kinases , as the cdc7 kinase functions in the parallel , mcm2 - 7 dependent branch . having each branch of the initiation pathway under the control of a protein kinase would thus afford tight regulation and coordination of the two branches , and perhaps explain why initiation requires two protein kinase activities . a model for the replication initiation pathway . the data presented here suggest two parallel pathways , one mcm2 - 7 dependent , the other mus101 dependent , which cooperate to load cdc45 at the g1/s transition . budding yeast dpb11 and fission yeast cut5 are considered homologues of one another because they are both required for dna replication , they are both required for replication checkpoint control , and they are both composed of reiterated brct domains . these arguments have also been extended to topbp1/mus101 , as it has been suggested that mus101 represents the metazoan homolog of dpb11/cut5 ( yamamoto et al . , 2000 ; the data reported here suggest a reconsideration of this designation , as we have shown that mus101 acts in a fundamentally different capacity than does dpb11 during the initiation of dna replication . mus101 is required for cdc45 loading , whereas dpb11 has been reported to act much later , after cdc45 has been loaded and , indeed , after the helicase has been activated and rpa has been recruited to the origin ( masumoto et al . , 2000 ) . another difference is that dpb11 function during initiation is tightly connected to pol  , as dpb11 was initially isolated as in a synthetic lethal screen for genes interacting with pol  , and dpb11 has since been shown to physically interact with the pol  complex . such interaction with pol epsilon is not likely to be the case for xmus101 , as it has been shown that cdc45 is required for pol  chromatin association in frog egg extracts ( mimura et al . , 2000 ) ; therefore , our data place xmus101 minimally two steps upstream of pol  in the initiation pathway . thus , it is likely that mus101 is not the metazoan orthologue of dpb11/cut5 , and that the dpb11/cut5 orthologue still awaits identification . blast searching of ncbi databases using drosophila mus101 sequences revealed several overlapping xenopus ests containing significant sequence homology to the fly mus101 gene . based on the sequence of the frog ests , primers were designed to amplify a 1,000 nucleotide fragment corresponding to nucleotides 3,5664,566 of the xmus101 open reading frame . the pcr fragment was then used as a probe to screen , by hybridization , a xenopus ovary cdna library . library screening resulted in the isolation of a full - length xmus101 cdna , which was then sequenced on both strands according to standard methods . pcr primers were designed to amplify fragments corresponding to the cooh - terminal 333 amino acids of xmus101 . the fragment was subcloned into the pet24a bacterial expression vector ( novagen ) and used to produce recombinant protein . six histidine - tagged recombinant protein was purified on a nickel agarose column , under native conditions . polyclonal antibodies were obtained , and affinity purified according to standard procedures ( harlow and lane , 1988 ) . antibodies against xenopus cdc45 , rpa , mcm10 , and cdc7 have been described ( walter and newport , 2000 ; walter , 2000 ; wohlschlegel et al . , 2002 ) . additionally , antibodies against xenopus mcm7 were obtained from john newport ( university of california san diego , san diego , ca ) , and against xenopus pol  and pol  from shou waga ( osaka university , osaka , japan ) . preparation of frog egg extracts , preparation of sperm chromatin , and dna replication analysis was all performed according to walter and newport ( 1999 ) . for immunodepletion , anti - xmus101 antibodies were coupled to 50 l of protein a sepharose beads , and then incubated with 200 l of frog egg extract on ice for 1 h. after incubation , the beads were pelleted , and the extract collected for an additional round of depletion . after the second round of depletion , the extract was used for the given experiment . for mock depletion , the procedure was identical except that anti - xmus101 antibodies were replaced by phosphate - buffered saline . orc depletion of npe was performed as described ( walter and newport , 2000 ) . for the experiments shown in fig . 2 , chromatin was isolated as described ( michael et al . , 2000 ) . 3 , chromatin isolation from npe was performed as described ( wohlschlegel et al . , 2002 ) . 4 , the chromatin was isolated according to jares and blow ( 2000 ) , using nuclear isolation buffer containing 100 mm kcl . blast searching of ncbi databases using drosophila mus101 sequences revealed several overlapping xenopus ests containing significant sequence homology to the fly mus101 gene . based on the sequence of the frog ests , primers were designed to amplify a 1,000 nucleotide fragment corresponding to nucleotides 3,5664,566 of the xmus101 open reading frame . the pcr fragment was then used as a probe to screen , by hybridization , a xenopus ovary cdna library . library screening resulted in the isolation of a full - length xmus101 cdna , which was then sequenced on both strands according to standard methods . pcr primers were designed to amplify fragments corresponding to the cooh - terminal 333 amino acids of xmus101 . the fragment was subcloned into the pet24a bacterial expression vector ( novagen ) and used to produce recombinant protein . six histidine - tagged recombinant protein was purified on a nickel agarose column , under native conditions . polyclonal antibodies were obtained , and affinity purified according to standard procedures ( harlow and lane , 1988 ) . antibodies against xenopus cdc45 , rpa , mcm10 , and cdc7 have been described ( walter and newport , 2000 ; walter , 2000 ; wohlschlegel et al . , 2002 ) . additionally , antibodies against xenopus mcm7 were obtained from john newport ( university of california san diego , san diego , ca ) , and against xenopus pol  and pol  from shou waga ( osaka university , osaka , japan ) . preparation of frog egg extracts , preparation of sperm chromatin , and dna replication analysis was all performed according to walter and newport ( 1999 ) . for immunodepletion , anti - xmus101 antibodies were coupled to 50 l of protein a sepharose beads , and then incubated with 200 l of frog egg extract on ice for 1 h. after incubation , the beads were pelleted , and the extract collected for an additional round of depletion . after the second round of depletion , the procedure was identical except that anti - xmus101 antibodies were replaced by phosphate - buffered saline . orc depletion of npe was performed as described ( walter and newport , 2000 ) . 2 , chromatin was isolated as described ( michael et al . , 2000 ) . 3 , chromatin isolation from npe was performed as described ( wohlschlegel et al . , 2002 ) . 4 , the chromatin was isolated according to jares and blow ( 2000 ) , using nuclear isolation buffer containing 100 mm kcl .
the initiation of eukaryotic dna replication involves origin recruitment and activation of the mcm2 - 7 complex , the putative replicative helicase . 
 mini - chromosome maintenance ( mcm)2 - 7 recruitment to origins in g1 requires origin recognition complex ( orc ) , cdt1 , and cdc6 , and activation at g1/s requires mcm10 and the protein kinases cdc7 and s - cdk , which together recruit cdc45 , a putative mcm2 - 7 cofactor required for origin unwinding . here 
 , we show that the xenopus brca1 cooh terminus repeat  containing xmus101 protein is required for loading of cdc45 onto the origin . 
 xmus101 chromatin association is dependent on orc , and independent of s - cdk and mcm2 - 7 . 
 these results define a new factor that is required for cdc45 loading . 
 additionally , these findings indicate that the initiation complex assembly pathway bifurcates early , after orc association with the origin , and that two parallel pathways , one controlled by mcm2 - 7 , and the other by xmus101 , cooperate to load cdc45 onto the origin .