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• W2022109078 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. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Hyrien O. Marheineke K. Goldar A. BioEssays. 2003; 25: 116-125Crossref PubMed Scopus (159) Google Scholar, 17Blow J.J. Gillespie P.J. Francis D. Jackson D.A. J. Cell Biol. 2001; 152: 15-26Crossref PubMed Scopus (125) Google Scholar). We found that replication initiates throughout S phase and at broadly distributed rather than strictly regular intervals, that interference between adjacent origins occurs, and that the frequency of initiation increases throughout S phase. We suggested that abundant potential origins may be defined by multiple Mcm complexes spread away from ORC rather than by ORC itself (13Lucas I. Chevrier-Miller M. Sogo J.M. Hyrien O. J. Mol. Biol. 2000; 296: 769-786Crossref PubMed Scopus (83) Google Scholar), so that a choice of which origins actually fire may occur during S phase to ensure an adequate distribution of initiation events (for a review, see Ref. 16Hyrien O. Marheineke K. Goldar A. BioEssays. 2003; 25: 116-125Crossref PubMed Scopus (159) Google Scholar). Indeed, Mcms bind DNA and initiate replication over a large region distant from ORC in egg extracts (18Edwards M.C. Tutter A.V. Cvetic C. Gilbert C.H. Prokhorova T.A. Walter J.C. J. Biol. Chem. 2002; 277: 33049-33057Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 19Harvey K.J. Newport J. Mol. Cell. Biol. 2003; 23: 6769-6779Crossref PubMed Scopus (46) Google Scholar). The mechanisms that control origin firing timing are still unclear. In budding yeast, where specific origins fire at specific times, the temporal program is established during the G1 phase (20Raghuraman M.K. Brewer B.J. Fangman W.L. Science. 1997; 276: 806-809Crossref PubMed Scopus (139) Google Scholar), and three kinases, Cdk1-Clb5, Mec1, and Rad53, seem implicated in its execution during S phase. Cdk1-Clb6 only activates early origins, whereas Cdk1-Clb5 activates both early and late origins (21Donaldson A.D. Raghuraman M.K. Friedman K.L. Cross F.R. Brewer B.J. Fangman W.L. Mol. Cell. 1998; 2: 173-182Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). A Mec1/Rad53-dependent checkpoint prevents the firing of late origins in the presence of stalled forks or DNA damage (22Santocanale C. Diffley J.F. Nature. 1998; 395: 615-618Crossref PubMed Scopus (533) Google Scholar, 23Shirahige K. Hori Y. Shiraishi K. Yamashita M. Takahashi K. Obuse C. Tsurimoto T. Yoshikawa H. Nature. 1998; 395: 618-621Crossref PubMed Scopus (359) Google Scholar). Interestingly, the Mec1/Rad53 pathway may also regulate origin firing time during unperturbed cell growth (23Shirahige K. Hori Y. Shiraishi K. Yamashita M. Takahashi K. Obuse C. Tsurimoto T. Yoshikawa H. Nature. 1998; 395: 618-621Crossref PubMed Scopus (359) Google Scholar, 24Sharma K. Weinberger M. Huberman J.A. Genetics. 2001; 159: 35-45PubMed Google Scholar). In animal cells, a correlation between replication timing, gene expression, and chromatin structure has long been observed (25Goren A. Cedar H. Nat. Rev. Mol. Cell. Biol. 2003; 4: 25-32Crossref PubMed Scopus (122) Google Scholar, 26McNairn A.J. Gilbert D.M. BioEssays. 2003; 25: 647-656Crossref PubMed Scopus (135) Google Scholar). The replication timing is established early in G1 phase when chromatin is repositioned in the nucleus after mitosis (27Dimitrova D.S. Gilbert D.M. Mol. Cell. 1999; 4: 983-993Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), and an intra-S checkpoint controls the timely assembly and disassembly of replication factories under conditions of replicational stress (28Dimitrova D.S. Gilbert D.M. Nat. Cell Biol. 2000; 2: 686-694Crossref PubMed Scopus (135) Google Scholar). In early Xenopus embryos, the lack of G1 phase, gene expression, and euchromatin/heterochromatin differentiation raises questions regarding the regulation and functional role of origin firing timing. We have previously shown by DNA combing that an intra S checkpoint regulates origin firing when sperm nuclei in egg extracts are challenged with aphidicolin, a DNA polymerase inhibitor (15Marheineke K. Hyrien O. J. Biol. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). We suggested that a checkpoint monitors the density of forks and regulates initiation to control the rate of DNA replication independently of the position of initiation events. Walter and Newport (29Walter J. Newport J.W. Science. 1997; 275: 993-995Crossref PubMed Scopus (144) Google Scholar) reported that when the concentration of sperm nuclei in egg extracts exceeds the concentration of cells in a Xenopus MBT embryo, the length of S phase increases, although the rate of replication fork movement appears unchanged. This might be due to an increase in replicon size, possibly by depletion of an initiation factor, or to staggered initiation at individual origins. The latter hypothesis was dismissed because the addition of the cyclin-dependent kinase inhibitor p21Cip1 after the start of S phase had little effect on subsequent replication (29Walter J. Newport J.W. Science. 1997; 275: 993-995Crossref PubMed Scopus (144) Google Scholar). However, it has since been clearly established that origins fire at different times through 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. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 16Hyrien O. Marheineke K. Goldar A. BioEssays. 2003; 25: 116-125Crossref PubMed Scopus (159) Google Scholar, 17Blow J.J. Gillespie P.J. Francis D. Jackson D.A. J. Cell Biol. 2001; 152: 15-26Crossref PubMed Scopus (125) Google Scholar). In this paper, we use molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts to directly examine the effect of the nucleocytoplasmic ratio and caffeine on the pattern of origin firing. Caffeine is a potent small molecule inhibitor of the kinase activity of the Mec1 homologs ATM and ATR in vitro (30Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar) and abrogates checkpoints in vivo (31Cortez D. J. Biol. Chem. 2003; 278: 37139-37145Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 32Kaufmann W.K. Heffernan T.P. Beaulieu L.M. Doherty S. Frank A.R. Zhou Y. Bryant M.F. Zhou T. Luche D.D. Noikolaishvilli-Feinberg N. Simpson D.A. Cordeiro-Stone M. Mutat Res. 2003; 532: 85-102Crossref PubMed Scopus (81) Google Scholar). We demonstrate that the longer S phase at high concentrations of nuclei is not due to a change in replicon size but to an expansion of the period of time over which origins fire and to a lower replication fork velocity. Treatment with caffeine increases origin firing but decreases fork velocity. Inhibition of ATR with a specific neutralizing antibody also increases origin firing. These results suggest that an intra-S checkpoint decreases origin firing in response to a limiting factor to ensure optimal fork progression. Replication of Sperm Nuclei in Xenopus Egg Extracts—Replication-competent extracts from unfertilized Xenopus eggs were prepared as described (33Blow J.J. Laskey R.A. Cell. 1986; 47: 577-587Abstract Full Text PDF PubMed Scopus (450) Google Scholar) and used fresh. Sperm nuclei (100–6000 nuclei/μl) were incubated in extracts in the presence of cycloheximide (250 μg/ml), energy mix (7.5 mm creatine phosphate, 1 mm ATP, 0.1 mm EGTA, pH 7.7, 1 mm MgCl2), 20 μm digoxigenin-dUTP (added at t = 0), and 20 μm biotin-dUTP (added at the indicated times) (Roche Applied Science). Replication was allowed to continue for 2 h. Aphidicolin block-and-release experiments were performed as described previously (15Marheineke K. Hyrien O. J. Biol. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Caffeine (or buffer alone as control) was added where indicated to a final concentration of 5 mm from a 100 mm solution freshly dissolved in 10 mm Pipes-NaOH, pH 7.4. ATR-neutralizing antibodies (34Lupardus P.J. Byun T. Yee M.C. Hekmat-Nejad M. Cimprich K.A. Genes Dev. 2002; 16: 2327-2332Crossref PubMed Scopus (144) Google Scholar) were added where indicated. Molecular Combing and Detection by Fluorescent Antibodies—DNA was extracted and combed as described (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. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Biotin was detected with Texas Red-conjugated antibodies (15Marheineke K. Hyrien O. J. Biol. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For the digoxigenin label, a mouse fluorescein isothiocyanate-conjugated anti-digoxigenin antibody was used followed by Alexa Fluor 488 rabbit anti-mouse and goat anti-rabbit antibodies (Molecular Probes, Inc., Eugene, OR) for enhancement. Measurements and Data Analysis—Images of the combed DNA molecules were acquired and measured as described (15Marheineke K. Hyrien O. J. Biol. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Fields of view were chosen at random in the fluorescein isothiocyanate channel and then photographed under the fluorescein isothiocyanate and the Texas Red filter. The size of most photographed molecules was 120 kb. All photographed fibers showed a continuous digoxigenin label, indicating complete replication. Replication eyes, gaps, and forks were defined as described under “Results.” Tracts of biotin-negative DNA needed to be >2 kb to be considered significant and scored as eyes. The replication extent of each fiber (or group of fibers) was defined as the sum of eye lengths divided by the total length of the molecule(s). Fork density is the total number of forks divided by total DNA length (kb) in each sample. The overall mean eye length and gap length were determined by dividing the total replicated or unreplicated length, respectively, by half the number of forks in each sample. Similarly, the overall mean eye-to-eye distance was determined by dividing the total DNA length by half the number of forks. Note that this is just twice the inverse of fork density. These numbers are not affected by the finite size of the analyzed molecules. However, to analyze the distribution of eye lengths, gap lengths, and eye-to-eye distances, incomplete eyes or gaps located on either end of each fiber had to be excluded. This bias resulted in a difference between “local” measurements and “overall” means calculated as above. Therefore, the mean value of “excluded” eye lengths, gap lengths, and eye-to-eye distances were also calculated by dividing the total excluded length of replicated, unreplicated, or total DNA, respectively, by half the number of excluded forks. The average fork velocity (kb/min) between two successive time points was calculated as the difference in replication extent between the two time points divided by the average fork density (number of forks/kb of total DNA) and by the time interval (min). The average fork density is defined as the mean of the fork density at the two time points. Small errors can therefore result if the change in fork density between the two time points is not linear. We also caution that with eyes of <2 kb being excluded from analysis, velocities may be slightly overestimated, but this should not affect comparisons between different concentrations of nuclei at similar replication extents. Alkaline-agarose Gel Electrophoresis—Sperm nuclei were incubated in fresh extracts added with 5 mm caffeine or buffer alone as control and one-fiftieth volume of [α-32P]dATP (3000 Ci/mmol). DNA was purified, separated on 1.1% alkaline-agarose gels, and analyzed as described (15Marheineke K. Hyrien O. J. Biol. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Shorter S Phase at Low Nuclei Concentration Is Due to More Synchronous Origin Firing and Faster Fork Progression— When sperm nuclei are incubated in Xenopus egg extracts, there is an initial lag period of ∼25 min during which replication origins are assembled on DNA and replication-competent nuclei are produced. After this lag, S phase starts abruptly. Most nuclei start to replicate within ∼5 min of each other. S phase is very brief (typically < 30 min), but its exact length depends on the concentration of nuclei. In order to visualize origin firing and fork progression at different nuclei concentrations, we used DNA combing, a powerful DNA spreading technique (35Michalet X. Ekong R. Fougerousse F. Rousseaux S. Schurra C. Hornigold N. van Slegtenhorst M. Wolfe J. Povey S. Beckmann J.S. Bensimon A. Science. 1997; 277: 1518-1523Crossref PubMed Scopus (518) Google Scholar) that has been adapted for single molecule analysis of DNA replication (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. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Sperm nuclei were incubated at 100 or 2000 nuclei/μl in egg extracts supplemented with digoxigenin-dUTP, so as to label all replicated DNA. Biotin-dUTP was added at different time points after the start of replication to differentially label early and late replicating sequences. After a 2-h incubation, the DNA was purified and combed on silanized coverslips, and the two labels were detected with fluorescent antibodies as described under “Experimental Procedures” (Fig. 1A). This double-labeling protocol allows us to ascertain the full replication of the fiber after 2 h and the fiber continuity between successive biotin-labeled tracts. Replication eyes are defined as regions replicated before the addition of biotin-dUTP and appear as tracts of digoxigenin-positive, biotin-negative DNA. Gaps are regions replicated after biotin-dUTP addition. Forks are the transition points between biotin-labeled and unlabeled segments. Eye-to-eye distances are measured between the midpoints of adjacent eyes. Combed DNA molecules are parallel, straight, and homogeneously stretched (1 μm = 2 kb) (Fig. 1A). About 50–80 randomly chosen DNA fibers (5–10 Mb) were analyzed for each time point. The replication extent (at the time of biotin-dUTP addition) of each group of fibers was defined as the sum of eye lengths divided by the total DNA length. The time required for >80% of the DNA to be replicated was shorter at 100 nuclei/μl (∼10 min) than at 2000 nuclei/μl (∼25 min) (Figs. 1D and 2A). This was also observed by measuring the time course of incorporation of α-32P]dATP into sperm DNA (not shown). The synchrony with which individual nuclei entered S phase was only 3–5 min tighter at 100 nuclei/μl than at 2000 nuclei/μl (as estimated by incorporation of rhodamine-dUTP into individual nuclei; not shown), which is insufficient to account for the 15-min difference in replication time. Therefore, S phase was about half as long at 100 nuclei/μl as at 2000 nuclei/μl. This may result from a faster progression of replication forks, a shorter interorigin distance, or a more synchronous firing of origins. It was shown using alkaline gel electrophoresis of nascent strands formed after release from Ara-C arrest that the rate of replication fork movement in Xenopus egg extracts is the same from 125 to 10,000 nuclei/μl (29Walter J. Newport J.W. Science. 1997; 275: 993-995Crossref PubMed Scopus (144) Google Scholar). However, fork velocity might be perturbed by Ara-C treatment, which is now known to uncouple helicase from polymerase movement (6Walter J. Newport J. Mol. Cell. 2000; 5: 617-627Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Therefore, we used DNA combing to monitor origin activation, measure interorigin distances, and estimate fork velocity at different stages of unperturbed S phase. Origin synchrony was assessed by determining the replication extent of each DNA fiber (sum of eye lengths divided by fiber length) and by plotting the lengths of individual eyes against the fiber's replication extent (Fig. 1, B and C). Each vertical alignment of dots in Fig. 1, B and C, thus represents the lengths of eyes of a single DNA fiber. A heterogeneous distribution of fiber replication extents and eye lengths was observed at all stages of S phase at 100 (Fig. 1B) and 2000 nuclei/μl (Fig. 1C). Small eyes, which indicate new initiation events, were visible at all time points and all replication extents. Thus, even at 100 nuclei/μl, replication origins fired continuously throughout S phase, as previously reported for plasmid DNA (13Lucas I. Chevrier-Miller M. Sogo J.M. Hyrien O. J. Mol. Biol. 2000; 296: 769-786Crossref PubMed Scopus (83) Google Scholar) or sperm nuclei at 2000 nuclei/μl (15Marheineke K. Hyrien O. J. Biol. Chem. 2001; 276: 17092-17100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Not only did origins within a given fiber fire asynchronously, but different fibers also showed a wide distribution of replication extent at all stages of S phase. To assess whether adjacent origins fire more synchronously than pairs of origins taken at random, the correlation coefficient between the lengths of adjacent eyes was calculated (excluding eyes >8 kb to avoid mergers). A weak but significant correlation of 0.16 was previously reported for a concentration of 3000 nuclei/μl, suggesting that origins are activated in clusters that fire at different times within S phase (17Blow J.J. Gillespie P.J. Francis D. Jackson D.A. J. Cell Biol. 2001; 152: 15-26Crossref PubMed Scopus (125) Google Scholar). We found similar correlations at all time points for both concentrations of nuclei (0.16–0.22; p < 0.05). We then had a closer look at fibers from the 30-min time point (Fig. 2, A and B). Both samples corresponded to very early S phase (replication extent r = 6.4% at 100 nuclei/μl and 2.2% at 2000 nuclei/μl). The distributions of the number of eyes per fiber (Fig. 2, C and D, black bars) were non-Poissonian (p < 0.001), with an excess of fibers containing either no eye or multiple eyes (compare black and white bars). This confirms that at either concentration of nuclei, origins are not activated independently of each other but as clusters. Fibers with no replication eye were 2.5-fold less frequent at 100 than at 2000 nuclei/μl (Fig. 2, A–D). The higher replication extent at 100 nuclei/μl was not due to a longer eye length (mean of 3.4 versus 3.0 kb) but to a higher fork density (total number of forks divided by total DNA length in each sample; 1/26.7 versus 1/70.2 kb). Extrapolation of the replication versus time plots (Fig. 1B) to 0% replication suggests that S phase started at the same time (t ∼27 min), and fork velocity was similar (∼1.1 kb·min–1; see “Experimental Procedures”) at the two nuclei concentrations. However, 2.6-fold more origins had fired at 100 than at 2000 nuclei/μl at 30 min. This was also observed with nuclei synchronized by aphidicolin block-and-release (see below) and therefore cannot be explained by an asynchrony with which nuclei initiate replication when the concentration of nuclei increases. Although the overall eye-to-eye distance (total DNA length divided by half the number of forks) was smaller at 100 than at 2000 nuclei/μl (53.4 versus 140.4 kb), the local eye-to-eye distances directly measured on individual fibers at 30 min were not markedly different (mean of 20.6 versus 24.7 kb) (Fig. 2A). This apparent discrepancy arises from the cluster organization and the limit in fiber length set by DNA breaks and/or microscope field. Incomplete eyes or gaps (and associated eye-to-eye distances) located on either fiber end are excluded from the individual measurements, and large (intercluster) distances have a greater probability to be excluded, whereas all DNA and forks are taken into account to calculate fork density (or overall eye-to-eye distance, which is twice the inverse of fork density). Importantly, it is possible to calculate the number and mean length of those excluded distances, as explained under “Experimental Procedures” (Fig. 2A). At 30 min, the excluded distances represented an important proportion (32 and 39%, respectively) of all eye-to-eye distances and were significantly shorter at 100 than at 2000 nuclei/μl (130 versus 319 kb). It is this class of distances (i.e. intercluster), not the local (intracluster) distances, that account for the large difference in fork density between the two samples. The analysis of gap lengths was entirely consistent with these conclusions (not further described, but see Fig. 2A). In summary, about 2 times more origin clusters (and 20–30% more origins per cluster) were activated at the start of S phase at 100 than at 2000 nuclei/μl. We next focused on the 37-min time points, which correspond to mid-S phase (r = 52.3% for 100 nuclei/μl and 30.0% for 2000 nuclei/μl). The overall (17.2 versus 18.6 kb), local (12.6 versus 14.2 kb; distributions shown on Fig. 2E), and excluded (42.3 versus 40.4 kb) eye-to-eye distances were now similar at both concentrations of nuclei. The higher replication extent at 100 nuclei/μl was entirely explained by a longer mean eye length (9.0 versus 5.6 kb). The 2.15-fold higher rate of eye growth was due to a higher rate of both fork progression and eye merger. Eyes of >25 kb were ∼10 times more frequent, and eyes of <5 kb were ∼25% less frequent at 100 nuclei/μl than at 2000 nuclei/μl, suggesting more mergers (not shown). Although fork velocity was only 1.3-fold higher at 100 nuclei/μl than at 2000 nuclei/μl (∼850 and ∼650 nt·min–1, respectively), replication from 30 to 37 min was 1.65-fold faster at 100 than at 2000 nuclei/μl (52.3–6.4 = 45.9% versus 30.0–2.2 = 27.8%). This implies that fork density increased faster at the beginning of S phase, although the same plateau was reached at 37 min (∼1 fork/9 kb). From 37 to 42 min, the fork density decreased from ∼1 fork/9 kb to ∼1 fork/14 kb at 100 nuclei/μl, consistent with mergers, but remained stable at ∼1 fork/9 kb at 2000 nuclei/μl (Fig. 2A). Despite the higher average fork density at 2000 than at 100 nuclei/μl, the replication extent increased 3.8-fold less (37.6–30.0 = 7.6% versus 81.3–52.3 = 29.0%). The replication extent at 42 min at 2000 nuclei/μl might have been underestimated, due to some bias in the fiber sample, because the plot in Fig. 1D shows an unexpected inflection at this point. Nevertheless, the fork velocity was ∼610 nt·min–1 at 100 nuclei/μl from 37 to 42 min, but only ∼420 nt·min–1 at 2000 nuclei/μl from 37 to 49 min. These data suggest that the rate of fork progression is more variable than hitherto appreciated (Fig. 2F). When replication reached >75–80%, replicon merge became predominant over new initiation. Eye-to-eye distances incr" @default.
• W2022109078 created "2016-06-24" @default.
• W2022109078 creator A5000672356 @default.
• W2022109078 creator A5007655086 @default.
• W2022109078 date "2004-07-01" @default.
• W2022109078 modified "2023-10-05" @default.
• W2022109078 title "Control of Replication Origin Density and Firing Time in Xenopus Egg Extracts" @default.
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