Analysis of Chromosome Replication with Eggs of Xenopus Laevis

Laskey, Ronald A.; Kearsey, Stephen; Méchali, Marcel

doi:10.1007/978-1-4684-4973-0_6

Cited by 17 publications

(19 citation statements)

References 45 publications

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“…1) 6,22 . To avoid the random gap problem, it has been argued that there must be some mechanism to coordinate origin firing so as to avoid large random gaps 22 .…”

Section: Introductionmentioning

confidence: 99%

“…1) 6,22 . To avoid the random gap problem, it has been argued that there must be some mechanism to coordinate origin firing so as to avoid large random gaps 22 . Surprisingly, in two cases in which the distribution of origin firing has been carefully examined -frog embryo extracts and fission yeast -the distribution was in fact random and large random gaps were observed 8,9,23,24 .…”

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confidence: 99%

“…Second, stochastic firing of origins leads to the so-called 'random gap' problemrandomly distributed origin firing will occasionally lead to large gaps between replication bubbles that would take an inordinately long time to replicate (Fig. 1) 6,22 . To avoid the random gap problem, it has been argued that there must be some mechanism to coordinate origin firing so as to avoid large random gaps 22 .…”

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confidence: 99%

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DNA replication timing: random thoughts about origin firing

2006

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Regions of metazoan genomes replicate at defined times within S phase. This observation suggests that replication origins fire with a defined timing pattern that remains the same from cycle to cycle. However, an alterative model based on the stochastic firing of origins may also explain replication timing. This model assumes varying origin efficiency instead of a strict origin-timing programme. Here, we discuss the evidence for both models.Models for the organization of eukaryotic DNA replication have to account for two diametrically opposed conceptions of replication. On one side of the spectrum is the replicon paradigm, exemplified by the mechanism of bacterial replication, in which origins are welldefined sites that fire once in each cell cycle, leading to uniform replication that is identical in every cell 1 . At the opposite end of the spectrum is the mechanism of replication in frog and fly embryos, in which replication initiates asynchronously throughout S phase at indiscriminate sites, presumably leading to a different, random pattern of replication in each cell 2,3 . It is clear that neither of these extreme mechanisms reflect the reality of replication in most eukaryotic somatic cells, but it is also clear that both models contain a kernel of truth. So how do we reconcile these apparently competing views of replication? Recent work suggests a way to bridge the gap and to reconcile stochastic origin firing with defined patterns of genome replication.It is useful to first establish which parts of each model generally apply to eukaryotic replication. The replicon paradigm, which serves as the foundation for most textbook models of eukaryotic replication, describes eukaryotic replication reasonably well, with two major exceptions: first, most eukaryotes do not seem to have well-defined, sequence-specific origins4 , 5. Consequently, it has been difficult to identify metazoan replication origins, and in the few cases in which they have been identified, they seem to have no particular sequence specificity. However, too few metazoan origins have been defined to exclude the possibility of origin specific sequences. The question of what constitutes a eukaryotic replication origin remains an active field of research, and as knowledge of the exact location of origins is not necessary for the ideas presented here, we will not pursue it further. Second, eukaryotic origins are inefficient and fire in only a subset of S phases 6 -only a fraction of the origins established in G1 are fired in S phase and it is a different set in each cell. In fission yeast, most origins fire about 30% of the time, whereas in mammals, the well-studied dihydrofolate reductase (DHFR) origins fire approximately 20% of the time 7, 8.In terms of these two exceptions to the replicon paradigm, eukaryotic replication looks more like the example of frog and fly embryos. Frog embryos go to the extreme of establishing origins randomly across the genome, without regard to DNA sequence 2,9 . This is not the case for eukaryotes in general, whi...

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“…1) 6,22 . To avoid the random gap problem, it has been argued that there must be some mechanism to coordinate origin firing so as to avoid large random gaps 22 .…”

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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DNA replication timing: random thoughts about origin firing

2006

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“…In Xenopus eggs, maternal histones are stored in large quantities in the cytoplasm [8]. The Xenopus egg extract system is competent for DNA replication, repair, and chromatin assembly.…”

Section: Introductionmentioning

confidence: 99%

Xenopus CENP-A assembly into chromatin requires base excision repair proteins

Zeitlin

Patel

Kavli³

et al. 2005

DNA Repair

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CENP-A is an essential histone H3 variant found in all eukaryotes examined to date. To begin to determine how CENP-A is assembled into chromatin, we developed a binding assay using sperm chromatin in cell-free extract derived from Xenopus eggs. Our data suggest that the catalytic activities of an unidentified deoxycytidine deaminase and UNG2, a uracil DNA glycosylase, are involved in CENP-A assembly. In support of this model, inhibiting deoxycytidine deaminase with zebularine, or uracil DNA glycosylase with Ugi, uracil or UTP results in a lack of detectable CENP-A on sperm DNA.Conversely, inducing DNA damage increases the level of CENP-A detected on sperm chromatin. Our data suggest that base excision repair may be involved in assembly of this histone H3 variant.3

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“…2,3 The problem is all the more acute in that early embryo cells lack an efficient S/M checkpoint, 4 which is used by many eukaryotic cells to delay entry into mitosis in the presence of unreplicated DNA. This problem is formally stated as the "random-completion problem," 5 and, because of the reasons explained above, its solution requires a mechanism that regulates replication other than sequence.…”

Section: Introductionmentioning

confidence: 99%

Persistence Length of Chromatin Determines Origin Spacing in Xenopus Early-Embryo DNA Replication: Quantitative Comparisons between Theory and Experiment

Jun

Herrick²,

Bensimon³

et al. 2004

Cell Cycle

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In Xenopus early embryos, replication origins neither require specific DNA sequences nor is there an efficient S/M checkpoint, even though the whole genome (3 billion bases) is completely duplicated within 10-20 minutes. This leads to the "random-completion problem" of DNA replication in embryos, where one needs to find a mechanism that ensures complete, faithful, timely reproduction of the genome without any sequence dependence of replication origins. We analyze recent DNA replication data in Xenopus laevis egg extracts and find discrepancies with models where replication origins are distributed independently of chromatin structure. Motivated by these discrepancies, we have investigated the role that chromatin looping may play in DNA replication. We find that the loop-size distribution predicted from a wormlike-chain model of chromatin can account for the spatial distribution of replication origins in this system quantitatively. Together with earlier findings of increasing frequency of origin firings, our results can explain the random-completion problem. The agreement between experimental data (molecular combing) and theoretical predictions suggests that the intrinsic stiffness of chromatin loops plays a fundamental biological role in DNA replication in early-embryo Xenopus in regulating the origin spacing.

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Analysis of Chromosome Replication with Eggs of Xenopus Laevis

Cited by 17 publications

References 45 publications

DNA replication timing: random thoughts about origin firing

DNA replication timing: random thoughts about origin firing

Xenopus CENP-A assembly into chromatin requires base excision repair proteins

Persistence Length of Chromatin Determines Origin Spacing in Xenopus Early-Embryo DNA Replication: Quantitative Comparisons between Theory and Experiment

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