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. 2019 Mar 12;116(11):4973-4982.
doi: 10.1073/pnas.1818680116. Epub 2019 Feb 4.

Dynamics of DNA Replication in a Eukaryotic Cell

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Free PMC article

Dynamics of DNA Replication in a Eukaryotic Cell

Thomas Kelly et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Each genomic locus in a eukaryotic cell has a distinct average time of replication during S phase that depends on the spatial and temporal pattern of replication initiation events. Replication timing can affect genomic integrity because late replication is associated with an increased mutation rate. For most eukaryotes, the features of the genome that specify the location and timing of initiation events are unknown. To investigate these features for the fission yeast, Schizosaccharomyces pombe, we developed an integrative model to analyze large single-molecule and global genomic datasets. The model provides an accurate description of the complex dynamics of S. pombe DNA replication at high resolution. We present evidence that there are many more potential initiation sites in the S. pombe genome than previously identified and that the distribution of these sites is primarily determined by two factors: the sequence preferences of the origin recognition complex (ORC), and the interference of transcription with the assembly or stability of prereplication complexes (pre-RCs). We suggest that in addition to directly interfering with initiation, transcription has driven the evolution of the binding properties of ORC in S. pombe and other eukaryotic species to target pre-RC assembly to regions of the genome that are less likely to be transcribed.

Keywords: DNA replication; replication dynamics; replication origins; replication timing; transcriptional interference.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Model for eukaryotic DNA replication. In step 1, ORC and associated initiation factors bind to sites in the genome in G1 phase and catalyze pre-RC assembly. The distribution of pre-RCs is described by a probability distribution giving the likelihood of stable assembly at each genomic position. A fixed number of pre-RCs per cell is assembled at sites chosen at random from this distribution. In step 2, assembled pre-RCs are fired at random at an increasing rate during S phase. Replication forks move bidirectionally from each initiation site at a constant rate. Any unfired pre-RCs encountered by forks are disassembled.
Fig. 2.
Fig. 2.
Simulation of DNA combing data. Simulations of S. pombe DNA replication based on the model described in Fig. 1 were performed with optimized firing parameters assuming an exponential distribution of the intervals between pre-RCs. The values of the parameters, optimized as described in Methods, were as follows: pre-RC density, 80 per Mb; rate of increase of firing probability per min, 0.022 events per min2 per pre-RC; maximum firing rate, 0.3 events per min per pre-RC; fork velocity, 2 kb/min. (A) Comparison of the observed density of replication forks with that predicted by the simulation. Each point represents a DNA molecule in the Kaykov and Nurse (21) dataset. The value on the ordinate is the observed density of forks in each molecule, and the value on the abscissa is the predicted density of forks for a molecule with the same percent replication. Slope of the regression line = 0.97; R2 = 0.63. (B) Comparison of the observed and predicted density of replication forks as a function of the fraction of the genome replicated. The observed fork densities (blue points) are the average observed values in windows of 5% replicated. The black line gives the fork densities predicted by the simulation. (C) Distributions of predicted and observed intercentroid distances [the distances between centers of replicated segments, referred to as interorigin distances in Kaykov and Nurse (21)]. The distributions are shown as semilog plots of the fraction of intercentroid distances greater than the value on the abscissa. Lower curves include 160 combed DNA molecules in the dataset of Kaykov and Nurse (21) with extents of replication ranging from 0 to 100%. Upper curves include a subset of molecules replicated from 0 to 10%.
Fig. 3.
Fig. 3.
Dynamics of S. pombe DNA replication. Simulation of S. pombe DNA replication with the optimized parameters given in the legend of Fig. 2. (A) Values of replication variables as a function of time during S phase. (B) Frequency distribution of the time required to complete DNA replication.
Fig. 4.
Fig. 4.
Simulation of polymerase usage. (A) Polymerase usage data for a segment of chromosome 2 from Daigaku et al. (26). Rightward fork frequency increases to the right at sites of initiation. Rightward fork frequency decreases to the right at sites of termination. (B) Initiations are excluded from transcribed regions. DeltaRF is defined as the change in rightward fork frequency in a 300-bp segment relative to that of the segment immediately to its left. Black line indicates deltaRF for all 300-bp segments (bins) in chromosome 2 as a function of AT content, blue line indicates deltaRF for 300-bp segments outside transcription units, red line indicates deltaRF for 300-bp segments inside transcription units, and blue dotted line indicates exponential fit. (C) Comparison of observed rightward fork frequencies in a segment of chromosome 2 (red, Watson strand; blue, Crick strand) with the rightward fork frequencies predicted by simulation of replication (black). The firing parameters for the simulation were the same as in Fig. 2. The relative probability of pre-RC assembly at each site in the genome, obtained as described in the text, was Pr(x)T(x)×e[21×AT(x)], where AT(x) is the fraction AT in a 25-bp window centered at position x and T(x) = 0 if the window overlaps a transcription unit, T(x) = 1 otherwise. (D) Comparison of observed rightward fork frequencies with rightward fork frequencies predicted by a simulation that disallows firing of pre-RCs at the Rif1 binding site (position 3380 kb). See text for details.
Fig. 5.
Fig. 5.
Replication fork directionality across S. pombe chromosome 2. The observed rightward fork frequencies in 300-bp segments of the 4.54-Mb chromosome 2 (red, Watson strand; blue, Crick strand) are from the polymerase usage data of Daigaku et al. (26). The predicted rightward fork frequencies (black line) are from a simulation with the parameters as described in the legend of Fig. 4. The simulation disallows firing of pre-RCs at six Rif1 sites in chromosome 2 (see text).
Fig. 6.
Fig. 6.
Prediction of replication timing. (A) Comparison of the observed replication timing profile of a segment of chromosome 2 with that predicted by simulation of DNA replication. The red line shows the copy number of each 1-kb segment of the genome obtained by deep sequencing of DNA from S phase cells [Sort-seq data of Daigaku et al. (26)]. This profile is taken as a measure, not necessarily linear, of the relative timing of replication of each segment. The predicted median times of replication of each 300-bp segment are from a simulation with the parameters described in Fig. 2. (B) Frequency distribution of predicted median replication times for all 300-bp segments of chromosome 2. (C) Time courses of replication of typical early and late replicating segments (from the positions indicated by arrows in A). The ordinate shows the fraction of cells that have replicated the segment at a given time.
Fig. 7.
Fig. 7.
Initiations and terminations. (A) Average number of initiations and terminations per cell cycle for each 300-bp segment in the same 500-kb region of chromosome 2 as in Figs. 4 and 6. The frequency of initiations and terminations in each segment of the genome was determined by simulations with a total of about 2M initiation events. The origins identified from polymerase usage data by Daigaku et al. (26) are shown below the figure with the height of each bar indicating the relative efficiency of use. (B) Probability of pre-RCs in 300-bp bins across chromosome 2 plotted as a cumulative distribution function (CDF). The probabilities were calculated from the function given in the legend of Fig. 4. The red dotted line is the CDF of a uniform distribution. Inset shows the local fluctuation in the probability of pre-RC assembly in a short segment of the chromosome. ST, subtelomere; CEN, centromere. (C) CDF of pre-RC site probability after elimination of centromere and subtelomeric regions, amounting to about 8% of the genome.

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