DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts

Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to progressively appear during early S phase and foci labelled early in one S phase colocalized with those labelled early in the next S phase. However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres. The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure. Replication initiated more frequently in the transcription unit than in the intergenic spacer. These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1–5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50–100 kb)

A Dynamic Stochastic Model for DNA Replication Initiation in Early Embryos

Background: Eukaryotic cells seem unable to monitor replication completion during normal S phase, yet must ensure a reliable replication completion time. This is an acute problem in early Xenopus embryos since DNA replication origins are located and activated stochastically, leading to the random completion problem. DNA combing, kinetic modelling and other studies using Xenopus egg extracts have suggested that potential origins are much more abundant than actual initiation events and that the time-dependent rate of initiation, I(t), markedly increases through S phase to ensure the rapid completion of unreplicated gaps and a narrow distribution of completion times. However, the molecular mechanism that underlies this increase has remained obscure.Methodology/Principal Findings: Using both previous and novel DNA combing data we have confirmed that I(t) increases through S phase but have also established that it progressively decreases before the end of S phase. To explore plausible biochemical scenarios that might explain these features, we have performed comparisons between numerical simulations and DNA combing data. Several simple models were tested: i) recycling of a limiting replication fork component from completed replicons; ii) time-dependent increase in origin efficiency; iii) time-dependent increase in availability of an initially limiting factor, e. g. by nuclear import. None of these potential mechanisms could on its own account for the data. We propose a model that combines time-dependent changes in availability of a replication factor and a fork-density dependent affinity of this factor for potential origins. This novel model quantitatively and robustly accounted for the observed changes in initiation rate and fork density.Conclusions/Significance: This work provides a refined temporal profile of replication initiation rates and a robust, dynamic model that quantitatively explains replication origin usage during early embryonic S phase. These results have significant implications for the organisation of replication origins in higher eukaryotes

Computational Methods to Study Kinetics of DNA Replication

New technologies such as DNA combing have led to the availability of large quanti-ties of data that describe the state of DNA while undergoing replication in S phase. In this chapter, we describe methods used to extract various parameters of replica-tion — fork velocity, origin initiation rate, fork density, numbers of potential and utilized origins — from such data. We first present a version of the technique that applies to “ideal ” data. We then show how to deal with a number of real-world complications, such as the asynchrony of starting times of a population of cells, the finite length of fragments used in the analysis, and the finite amount of DNA in a chromosome. Key words: DNA replication, replication fork velocity, origin initiation

How Xenopus laevis embryos replicate reliably: investigating the random-completion problem

DNA synthesis in \textit{Xenopus} frog embryos initiates stochastically in time at many sites (origins) along the chromosome. Stochastic initiation implies fluctuations in the time to complete and may lead to cell death if replication takes longer than the cell cycle time ($\approx 25$ min). Surprisingly, although the typical replication time is about 20 min, \textit{in vivo} experiments show that replication fails to complete only about 1 in 300 times. How is replication timing accurately controlled despite the stochasticity? Biologists have proposed two solutions to this "random-completion problem." The first solution uses randomly located origins but increases their rate of initiation as S phase proceeds, while the second uses regularly spaced origins. In this paper, we investigate the random-completion problem using a type of model first developed to describe the kinetics of first-order phase transitions. Using methods from the field of extreme-value statistics, we derive the distribution of replication-completion times for a finite genome. We then argue that the biologists' first solution to the problem is not only consistent with experiment but also nearly optimizes the use of replicative proteins. We also show that spatial regularity in origin placement does not alter significantly the distribution of replication times and, thus, is not needed for the control of replication timing.Comment: 16 pages, 9 figures, submitted to Physical Review

Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics

Reconstruction of cell population dynamics using CFSE

Background: Quantifying cell division and death is central to many studies in the biological sciences. The fluorescent dye CFSE allows the tracking of cell division in vitro and in vivo and provides a rich source of information with which to test models of cell kinetics. Cell division and death have a stochastic component at the single-cell level, and the probabilities of these occurring in any given time interval may also undergo systematic variation at a population level. This gives rise to heterogeneity in proliferating cell populations. Branching processes provide a natural means of describing this behaviour. Results: We present a likelihood-based method for estimating the parameters of branching process models of cell kinetics using CFSE-labeling experiments, and demonstrate its validity using synthetic and experimental datasets. Performing inference and model comparison with real CFSE data presents some statistical problems and we suggest methods of dealing with them. Conclusion: The approach we describe here can be used to recover the (potentially variable) division and death rates of any cell population for which division tracking information is available

RsaI repetitive DNA in Buffalo Bubalus bubalis representing retrotransposons, conserved in bovids, are part of the functional genes

<p>Abstract</p> <p>Background</p> <p>Repetitive sequences are the major components of the eukaryotic genomes. Association of these repeats with transcribing sequences and their regulation in buffalo <it>Bubalus bubalis </it>has remained largely unresolved.</p> <p>Results</p> <p>We cloned and sequenced <it>RsaI </it>repeat fragments pDp1, pDp2, pDp3, pDp4 of 1331, 651, 603 and 339 base pairs, respectively from the buffalo, <it>Bubalus bubalis</it>. Upon characterization, these fragments were found to represent retrotransposons and part of some functional genes. The resultant clones showed cross hybridization only with buffalo, cattle, goat and sheep genomic DNA. Real Time PCR, detected ~2 × 10⁴copies of pDp1, ~ 3000 copies of pDp2 and pDp3 and ~ 1000 of pDp4 in buffalo, cattle, goat and sheep genomes, respectively. <it>RsaI </it>repeats are transcriptionally active in somatic tissues and spermatozoa. Accordingly, pDp1 showed maximum expression in lung, pDp2 and pDp3 both in Kidney, and pDp4 in ovary. Fluorescence <it>in situ </it>hybridization showed repeats to be distributed all across the chromosomes.</p> <p>Conclusions</p> <p>The data suggest that <it>RsaI </it>repeats have been incorporated into the exonic regions of various transcribing genes, possibly contributing towards the architecture and evolution of the buffalo and related genomes. Prospects of our present work in the context of comparative and functional genomics are highlighted.</p

Stochastic Models of Lymphocyte Proliferation and Death

Quantitative understanding of the kinetics of lymphocyte proliferation and death upon activation with an antigen is crucial for elucidating factors determining the magnitude, duration and efficiency of the immune response. Recent advances in quantitative experimental techniques, in particular intracellular labeling and multi-channel flow cytometry, allow one to measure the population structure of proliferating and dying lymphocytes for several generations with high precision. These new experimental techniques require novel quantitative methods of analysis. We review several recent mathematical approaches used to describe and analyze cell proliferation data. Using a rigorous mathematical framework, we show that two commonly used models that are based on the theories of age-structured cell populations and of branching processes, are mathematically identical. We provide several simple analytical solutions for a model in which the distribution of inter-division times follows a gamma distribution and show that this model can fit both simulated and experimental data. We also show that the estimates of some critical kinetic parameters, such as the average inter-division time, obtained by fitting models to data may depend on the assumed distribution of inter-division times, highlighting the challenges in quantitative understanding of cell kinetics

The Dyad Symmetry Element of Epstein-Barr Virus Is a Dominant but Dispensable Replication Origin

OriP, the latent origin of Epstein-Barr virus (EBV), consists of two essential elements: the dyad symmetry (DS) and the family of repeats (FR). The function of these elements has been predominantly analyzed in plasmids transfected into transformed cells. Here, we examined the molecular functions of DS in its native genomic context and at an ectopic position in the mini-EBV episome. Mini-EBV plasmids contain 41% of the EBV genome including all information required for the proliferation of human B cells. Both FR and DS function independently of their genomic context. We show that DS is the most active origin of replication present in the mini-EBV genome regardless of its location, and it is characterized by the binding of the origin recognition complex (ORC) allowing subsequent replication initiation. Surprisingly, the integrity of oriP is not required for the formation of the pre-replicative complex (pre-RC) at or near DS. In addition we show that initiation events occurring at sites other than the DS are also limited to once per cell cycle and that they are ORC-dependent. The deletion of DS increases initiation from alternative origins, which are normally used very infrequently in the mini-EBV genome. The sequence-independent distribution of ORC-binding, pre-RC-assembly, and initiation patterns indicates that a large number of silent origins are present in the mini-EBV genome. We conclude that, in mini-EBV genomes lacking the DS element, the absence of a strong ORC binding site results in an increase of ORC binding at dispersed sites