Control of Replication Origin Density and Firing Time in Xenopus Egg Extracts

Abstract

A strict control of replication origin density and firing time is essential to chromosomal stability. Replication origins in early frog embryos are located at apparently random sequences, are spaced at close (∼10-kb) intervals, and are activated in clusters that fire at different times throughout a very brief S phase. Using molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts, we show that the temporal order of origin firing can be modulated by the nucleocytoplasmic ratio and the checkpoint-abrogating agent caffeine in the absence of external challenge. Increasing the concentration of nuclei in the extract increases S phase length. Contrary to a previous interpretation, this does not result from a change in local origin spacing but from a spreading of the time over which distinct origin clusters fire and from a decrease in replication fork velocity. Caffeine addition or ATR inhibition with a specific neutralizing antibody increases origin firing early in S phase, suggesting that a checkpoint controls the time of origin firing during unperturbed S phase. Furthermore, fork progression is impaired when excess forks are assembled after caffeine treatment. We also show that caffeine allows more early origin firing with low levels of aphidicolin treatment but not higher levels. We propose that a caffeine-sensitive, ATR-dependent checkpoint adjusts the frequency of initiation to the supply of replication factors and optimizes fork density for safe and efficient chromosomal replication during normal S phase. A strict control of replication origin density and firing time is essential to chromosomal stability. Replication origins in early frog embryos are located at apparently random sequences, are spaced at close (∼10-kb) intervals, and are activated in clusters that fire at different times throughout a very brief S phase. Using molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts, we show that the temporal order of origin firing can be modulated by the nucleocytoplasmic ratio and the checkpoint-abrogating agent caffeine in the absence of external challenge. Increasing the concentration of nuclei in the extract increases S phase length. Contrary to a previous interpretation, this does not result from a change in local origin spacing but from a spreading of the time over which distinct origin clusters fire and from a decrease in replication fork velocity. Caffeine addition or ATR inhibition with a specific neutralizing antibody increases origin firing early in S phase, suggesting that a checkpoint controls the time of origin firing during unperturbed S phase. Furthermore, fork progression is impaired when excess forks are assembled after caffeine treatment. We also show that caffeine allows more early origin firing with low levels of aphidicolin treatment but not higher levels. We propose that a caffeine-sensitive, ATR-dependent checkpoint adjusts the frequency of initiation to the supply of replication factors and optimizes fork density for safe and efficient chromosomal replication during normal S phase. A strict control of replication origin density and time of activation is required to ensure that no DNA stretch is left unreplicated at the end of S phase. Replication initiation is governed by a conserved pathway of protein interactions at DNA replication origins (1Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1394) Google Scholar). During late mitosis and the G1 phase, prereplicative complexes are formed at sites defined by ORC, a six-subunit protein complex that directs the loading of other prereplicative complex components, including Cdc6, Cdt1, and the Mcm2-7 complex (2Romanowski P. Madine M.A. Rowles A. Blow J.J. Laskey R.A. Curr. Biol. 1996; 6: 1416-1425Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 3Coleman T.R. Carpenter P.B. Dunphy W.G. Cell. 1996; 87: 53-63Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 4Rowles A. Chong J.P. Brown L. Howell M. Evan G.I. Blow J.J. Cell. 1996; 87: 287-296Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 5Maiorano D. Moreau J. Mechali M. Nature. 2000; 404: 622-625Crossref PubMed Scopus (295) Google Scholar). After the G1/S transition, the prereplicative complex is converted to a preinitiation complex. This process is triggered by at least two kinases, Cdc7/Dbf4 and the S-cyclin-dependent kinases, and involves the ordered binding of numerous factors that ultimately unwind origin DNA and recruit DNA polymerases (6Walter J. Newport J. Mol. Cell. 2000; 5: 617-627Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 7Kubota Y. Takase Y. Komori Y. Hashimoto Y. Arata T. Kamimura Y. Araki H. Takisawa H. Genes Dev. 2003; 17: 1141-1152Crossref PubMed Scopus (169) Google Scholar). In early Xenopus embryos, S phase is very brief (∼20 min), and replication initiates without sequence specificity and at close intervals (∼10 kb) (8Hyrien O. Méchali M. EMBO J. 1993; 12: 4511-4520Crossref PubMed Scopus (171) Google Scholar). Site-specific initiation is only detected after the midblastula transition (MBT), 1The abbreviations used are: MBT, midblastula transition; Pipes, 1,4-piperazinediethanesulfonic acid. when transcription resumes (9Hyrien O. Maric C. Méchali M. Science. 1995; 270: 994-997Crossref PubMed Scopus (275) Google Scholar). Replicon size increases slightly at the MBT and more significantly at later stages (9Hyrien O. Maric C. Méchali M. Science. 1995; 270: 994-997Crossref PubMed Scopus (275) Google Scholar, 10Maric C. Levacher B. Hyrien O. J. Mol. Biol. 1999; 291: 775-788Crossref PubMed Scopus (19) Google Scholar). The mechanisms regulating these changes are unknown, but one clue is that the MBT occurs after a critical number of nuclei accumulate in the embryo (11Newport J. Kirschner M. Cell. 1982; 30: 675-686Abstract Full Text PDF PubMed Scopus (1183) Google Scholar). A completely random distribution of origins would generate some unacceptably large interorigin distances in the early Xenopus embryo (12Laskey R.A. J. Embryol. Exp. Morphol. 1985; 89: 285-296PubMed Google Scholar). To understand the mechanisms that ensure complete chromosome replication, we and others have studied the distribution of initiation events on single DNA molecules of plasmid and sperm nuclei replicating in egg extracts (13Lucas I. Chevrier-Miller M. Sogo J.M. Hyrien O. J. Mol. Biol. 2000; 296: 769-786Crossref PubMed Scopus (83) Google Scholar, 14Herrick J. Stanislawski P. Hyrien O. Bensimon A. J. Mol. Biol. 2000; 300: 1133-1142Crossref PubMed Scopus (96) Google Scholar, 15Marheineke K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, O. K. A. 2003; PubMed Scopus Google Scholar, J.J. D. J. Biol. PubMed Scopus Google Scholar). We that replication initiates throughout S phase and at intervals, that origins and that the frequency of initiation increases throughout S phase. We that origins be defined by complexes from by (13Lucas I. Chevrier-Miller M. Sogo J.M. Hyrien O. J. Mol. Biol. 2000; 296: 769-786Crossref PubMed Scopus (83) Google that a of which origins fire during S phase to ensure distribution of initiation events a O. K. A. 2003; PubMed Scopus Google Scholar). DNA and replication over a large from in egg extracts C. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Newport J. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). The mechanisms that control origin firing are In specific origins fire at specific the temporal is during the G1 phase Science. PubMed Scopus Google and kinases, and in during S phase. only early early and late origins Mol. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). A checkpoint the firing of late origins in the of forks or DNA C. Nature. PubMed Scopus Google Scholar, K. Y. K. M. K. C. T. H. Nature. PubMed Scopus Google Scholar). the pathway also origin firing time during unperturbed K. Y. K. M. K. C. T. H. Nature. PubMed Scopus Google Scholar, K. M. Google Scholar). In a replication and A. H. Rev. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google Scholar). The replication is early in G1 phase when is in the after mitosis Mol. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google and checkpoint controls the and of replication of Biol. 2000; PubMed Scopus Google Scholar). In early Xenopus embryos, the of G1 phase, and the and of origin firing We have by DNA combing that S checkpoint origin firing when sperm nuclei in egg extracts are with a DNA K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We that a checkpoint the density of forks and initiation to control the of DNA replication of the of initiation and Newport J. Newport Science. PubMed Scopus Google that when the concentration of sperm nuclei in egg extracts the concentration of in a Xenopus MBT the of S phase the of replication fork This be to in by of initiation or to initiation at The the addition of the after the of S phase on replication J. Newport Science. PubMed Scopus Google Scholar). that origins fire at different times S phase in egg extracts (13Lucas I. Chevrier-Miller M. Sogo J.M. Hyrien O. J. Mol. Biol. 2000; 296: 769-786Crossref PubMed Scopus (83) Google Scholar, 14Herrick J. Stanislawski P. Hyrien O. Bensimon A. J. Mol. Biol. 2000; 300: 1133-1142Crossref PubMed Scopus (96) Google Scholar, 15Marheineke K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, O. K. A. 2003; PubMed Scopus Google Scholar, J.J. D. J. Biol. PubMed Scopus Google Scholar). In this we molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts to the of the nucleocytoplasmic ratio and caffeine on the of origin Caffeine is a of the of the and ATR in P. Y. 1999; Google and in D. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. T. M. 2003; PubMed Scopus Google Scholar). We that the S phase at of nuclei is not to a change in size but to of the of time over which origins fire and to a replication fork velocity. with caffeine increases origin firing but fork velocity. of ATR with a specific neutralizing antibody also increases origin that checkpoint origin firing in to a to ensure fork Replication of in Xenopus extracts from Xenopus J.J. Laskey R.A. Cell. Full Text PDF PubMed Scopus Google and used nuclei in extracts in the of at and at the Replication to for K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Caffeine to a concentration of from a in T. M. Genes Dev. 2002; PubMed Scopus Google and by and J. Stanislawski P. Hyrien O. Bensimon A. J. Mol. Biol. 2000; 300: 1133-1142Crossref PubMed Scopus (96) Google Scholar, 15Marheineke K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). detected with K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the a antibody used by and for and of the DNA molecules and K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of at random in the and the and the The size of molecules a complete Replication and forks defined of DNA to be to be and The replication of of defined the of by the of the density is the number of forks by DNA in The and by the or unreplicated by the number of forks in the by the DNA by the number of that this is the of fork are not by the size of the to the distribution of and or located on end of to be This in a and the of and distances also by the of or by the number of The fork two time the in replication the two time by the fork density of of and by the time The fork density is defined the of the fork density at the two time can result the change in fork density the two time is not We also that with of from be slightly but this not different of nuclei at replication nuclei in extracts with caffeine or control and of DNA on and K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). S at to and sperm nuclei are in Xenopus egg extracts, is of during which replication origins are assembled on DNA and nuclei are After this S phase nuclei to of S phase is very brief min), but on the concentration of In order to origin firing and fork progression at different nuclei we used DNA a DNA spreading C. M. J. Bensimon A. Science. PubMed Scopus Google that for single of DNA replication J. Stanislawski P. Hyrien O. Bensimon A. J. Mol. Biol. 2000; 300: 1133-1142Crossref PubMed Scopus (96) Google Scholar, 15Marheineke K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). nuclei at or in egg extracts with to at different time after the of replication to early and late replicating After a the DNA and on and the two detected with This allows to the replication of the after and the Replication are defined the addition of and of are after are the transition and distances are the of DNA molecules are and kb) DNA for time The replication the time of of of defined the of by the DNA length. The time required for of the DNA to be at (∼10 at and This also by the time of of sperm DNA The with which nuclei S phase only at at by of not which is to for the in replication S phase at at This result from a progression of replication a interorigin or a more firing of of formed after from that the of replication fork in Xenopus egg extracts is the from to J. Newport Science. PubMed Scopus Google Scholar). fork be by which is to from (6Walter J. Newport J. Mol. Cell. 2000; 5: 617-627Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). we used DNA combing to origin interorigin and fork at different stages of unperturbed S phase. by the replication of DNA of by and by the of the replication and of in and the of of a single DNA A distribution of replication and at stages of S phase at and which initiation at time and replication at replication origins throughout S phase, for plasmid DNA (13Lucas I. Chevrier-Miller M. Sogo J.M. Hyrien O. J. Mol. Biol. 2000; 296: 769-786Crossref PubMed Scopus (83) Google or sperm nuclei at K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). only origins a fire but different also a distribution of replication at stages of S phase. To origins fire more of origins at the the of to A but of for a concentration of suggesting that origins are activated in clusters that fire at different times S phase J.J. D. J. Biol. PubMed Scopus Google Scholar). We at time for of nuclei We a at from the time A and to very early S phase at and at The of the number of and with excess of no or and This that at concentration of origins are not activated of other but with no replication at at The higher replication at not to a of kb) but to a higher fork density number of forks by DNA in of the replication time to replication that S phase at the time min), and fork at the two nuclei more origins at at at This also with nuclei by aphidicolin and be by with which nuclei replication when the concentration of nuclei the DNA by the number of at at the local distances on at not different of kb) This from the and the in by DNA or located on end are from the and large distances have a to be DNA and forks are to fork density which is the of fork is to the number and of the distances and of distances and significantly at at is this of distances not the local that for the large in fork density the two The of with these but In times more origin clusters more origins activated at the of S phase at at We on the time which to phase for and for The local on and kb) distances at of The higher replication at by a The higher of to a higher of fork progression and of times more and of at at suggesting more fork only higher at at and replication from to at at This that fork density at the of S phase, the at to the fork density from to at with but at at the higher fork density at at the replication The replication at at have to some in the the in at this the fork at from to but only at from to that the of fork progression is more replication over distances and unreplicated in a of and of a large of origin clusters in late S phase. 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In the of the nucleocytoplasmic ratio from to and from to To the that these are not to a different with which nuclei S phase at different we nuclei at or in the of aphidicolin and these the nuclei DNA replication of other K. Hyrien O. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). DNA combing the in S phase and temporal order of origin firing with nuclei at nuclei with the after from J. Newport Science. PubMed Scopus Google but significantly during unperturbed S phase Caffeine the of Replication of ATR-dependent order to the of the replication checkpoint in normal S phase we egg extracts for with of the ATR and P. Y. 1999; Google Scholar, D. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. T. M. 2003; PubMed Scopus Google sperm nuclei at or at in the of The at and the of DNA replication by and A and Caffeine the of early in S phase by at A but at progression at this size We that caffeine increases origin firing at the of S phase, at nuclei This by combing DNA from sperm nuclei in the or absence of caffeine and in S phase and the replication higher in the of caffeine in the control This from distances of kb) and only slightly of with the of the in replication and at caffeine the of DNA during early S phase, this that no of caffeine on S phase the fork density higher in the of caffeine throughout S phase The in the frequency of initiation early in S phase in a spacing of clusters of of unreplicated a spacing of clusters at spacing in the of caffeine in the control at kb) and A excess of distances in the and not of at from to to a size in the of caffeine kb) in the control This not result from a different frequency of spacing in fork throughout S phase in the of caffeine late in S phase in the in some but not in the caffeine Furthermore, the more in the of caffeine to kb) in the control to kb) from to the fork at that time these that a of the forks be in the We that initiation during S phase is control of a checkpoint in Xenopus egg This control is at nuclei suggesting that of some increases the checkpoint of this control by caffeine in initiation and or of some forks late in S phase. of caffeine at We these also when nuclei from a replication with aphidicolin extract with or without the replication higher in the of caffeine in the control to distances kb) and slightly spacing of of unreplicated but spacing clusters the in replication and spacing low and without or with caffeine that replication forks after aphidicolin and that this is not by checkpoint also that caffeine increases initiation early in S phase. Caffeine is of and ATR checkpoint is activated by DNA ATR binding and are by replication forks L. Science. 2003; 300: PubMed Scopus Google Scholar). Furthermore, ATR with during unperturbed DNA replication in Xenopus M. Newport Curr. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). To ATR controls origin firing during unperturbed S phase, we the of T. M. Genes Dev. 2002; PubMed Scopus Google on by with caffeine A and the addition of the of early in S phase to a size we that ATR inhibition origin This result that the checkpoint that during unperturbed S phase is at least in on ATR We to this checkpoint is to the one that origin firing in to In a previous K. Hyrien O. J. Biol. 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The size at aphidicolin in the of but this not in other that initiation is control of a checkpoint at The inhibition of DNA by aphidicolin a of initiation by caffeine more to are in the absence of a this higher concentration of aphidicolin a different or checkpoint that We have origin firing and S phase in Xenopus egg This that the replication can be modulated by the nucleocytoplasmic ratio and the checkpoint of origins are activated in clusters that fire at different Increasing the concentration of nuclei in the extract does not origin the time over which distinct clusters and the progression of replication The two S phase is at concentration of Caffeine the spreading of origin firing at nuclei a checkpoint that to the nucleocytoplasmic in the absence of replication or controls the time at which different origin clusters caffeine does not fork and at least some forks to or late in S phase. ATR inhibition with specific to caffeine on origin a for ATR in checkpoint during unperturbed S phase. to be clusters is a of replication origins 2000; PubMed Scopus Google Scholar). The Xenopus clusters origins and J.J. D. J. Biol. PubMed Scopus Google Scholar). we show this is at of but different clusters fire over a time at low This not when the the of not significantly change with nuclei concentration This is of and not are not by this In the distribution of number DNA and combing of the number or of replication which essential to this The of origin firing a be in two some distinct from origin firing of a DNA the of initiation that the firing of a origin the of kb) initiation We that a origin to a replication the of origins to the of spaced origins kb) would be by the of DNA (13Lucas I. Chevrier-Miller M. Sogo J.M. Hyrien O. J. Mol. Biol. 2000; 296: 769-786Crossref PubMed Scopus (83) Google of the of size of K. Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar). of a DNA to a single replication the of and the of origins J. Bensimon A. J. PubMed Scopus Google Scholar). The in the of or DNA Replication that the concentration of nuclei in egg extract S phase J. Newport Science. PubMed Scopus Google Scholar). and Newport J. Newport Science. PubMed Scopus Google that this only occurs a concentration of we a and This be by in extract or by the that and Newport used to A to embryo the MBT only occurs at the In S phase to the MBT this be in Xenopus and Newport J. Newport Science. 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The fork density is of concentration but can be by caffeine treatment. Caffeine and ATR in P. Y. 1999; Google Scholar). is activated in to DNA ATR in complex with to of DNA at replication forks L. Science. 2003; 300: PubMed Scopus Google Scholar). only a low of DNA is with unperturbed replication ATR with during unperturbed DNA replication in Xenopus M. Newport Curr. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the in fork density early in S phase ATR to origin firing and fork that origin firing in S phase this that a caffeine-sensitive, checkpoint also and origins in the absence of DNA H. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of Cdc7/Dbf4 or activation checkpoint inhibition of origin firing K. M. D. J. Mol. Cell. 2000; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. J. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the that origin firing to a caffeine that other also be Caffeine are not to In caffeine to in and to by with of D. J. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. T. M. 2003; PubMed Scopus Google Scholar). In Xenopus egg extracts, caffeine does ATR-dependent events T. M. Genes Dev. 2002; PubMed Scopus Google Scholar). of caffeine is the pathway K. J. A. Rev. 1999; Google Scholar). we that a PubMed Scopus Google does not the of in egg extracts not the of caffeine on origin firing is to be to and Newport J. Newport Science. PubMed Scopus Google that the to nuclei from on DNA a of addition to nuclei from aphidicolin Howell M. T. Blow J.J. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google with late We have by DNA combing the of the or to nuclei from aphidicolin not A decrease in replication and fork density throughout S phase, suggesting inhibition of late This and Newport not a of In we that phase checkpoint controls the time of origin firing in Xenopus egg extracts, early replication is from and The replication can be that origins are activated at frequency S phase and origin the of initiation over distances and in of replication and the frequency of initiation is control of a caffeine-sensitive, ATR-dependent is no to a of early and late replicating in this We that the checkpoint adjusts the frequency of and the of DNA by in to replication fork and replication forks throughout S phase. is to the and of the checkpoint and to the replication and of and transcription A. H. Rev. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar, 2003; PubMed Scopus Google later in on this replication We B. and C. for P. and K. for the and J. J. and the for critical of the