Do replication forks control late origin firing in Saccharomyces cerevisiae?

Abstract

Recent studies of eukaryotic DNA replication timing profiles suggest that the time-dependent rate of origin firing, I(t), has a universal shape, which ensures a reproducible replication completion time. However, measurements of I(t) are based on population averages, which may bias the shape of the I(t) because of imperfect cell synchrony and cell-to-cell variability. Here, we measure the population-averaged I(t) profile from synchronized Saccharomyces cerevisiae cells using DNA combing and we extract the single-cell I(t) profile using numerical deconvolution. The single cell I(t) and the population-averaged I(t) extracted from DNA combing and replication timing profiles are similar, indicating a genome scale invariance of the replication process, and excluding cell-to-cell variability in replication time as an explanation for the shape of I(t). The single cell I(t) correlates with fork density in wild-type cells, which is specifically loosened in late S phase in the clb5Δ mutant. A previously proposed numerical model that reproduces the wild-type I(t) profile, could also describe the clb5Δ mutant I(t) once modified to incorporate the decline in CDK activity and the looser dependency of initiation on fork density in the absence of Clb5p. Overall, these results suggest that the replication forks emanating from early fired origins facilitate origin firing in later-replicating regions.

Figure 1.

Experimental workflow and flow cytometry analysis of DNA content following α-factor release. (A) Cells were synchronized in G1 with α-factor and synchronously released into S phase by washing out the α-factor and transferring in fresh medium containing BrdU and nocodazole. Samples were collected every 15 min over a 150-min period, at which time the cells reached the G2/M phase. An aliquot of each timed sample was used to monitor S phase progression by flow cytometry fluorescence-activated cell sorter. The remaining part of the timed sample was transferred to fresh medium containing nocodazole and thymidine at 30°C up to 150 min. Each sample was divided in two parts, one was analysed by DNA combing and the other part was used to quantify BrdU incorporation. (B) Experimental data (open symbols) are fitted to equation (1) using a simplex algorithm (solid line, χ²= 1.03 and P < 10⁻⁴). (C) Linear correlation coefficient between two consecutive flow cytometry profiles. The linear correlation coefficient, R, was calculated between profiles measured at time n−1 and time n. The time n was assigned to the obtained value of R. The flow cytometry profile at time n = 0 was compared with itself and therefore R = 1. The starting time (t_start = 30 min) and the length of S phase (T_S = 42 ± 15 min) were measured by interpolating the obtained profile to a Gaussian function.

Figure 2.

Analysis of S phase parameters in the same cell population as in Figure 1. (A) Fraction of replicated DNA, f_DNA(t), as a function of time. Data points were extracted by measuring the amount of incorporated BrdU as described in ‘Materials and Methods’ section (open circle). The origin of time axis was set to the starting time of the S phase, t_start, which is 30 min after G1 release (time = t−t_start). To show that f_DNA(t) follows a sigmoidal shape (8), it was fitted to a Boltzmann sigmoidal function (solid black line, χ²= 1.003 and P < 10⁻⁴). (B) Total rate of DNA synthesis as a function of average DNA content. (C) Time-dependent distribution of DNA content (time = t−t_start). Inset: variation of the distributions width as a function of time (time = t−t_start). (D) Probability, n(t), for a cell to be in S phase as a function of time (time = t−t_start) calculated as described in ‘Material and Methods’ section. The black dashed line is a guide for the eye.

Figure 3.

Examples of combed DNA fibres from the same cell population as in Figures 1 and 2. BrdU incorporation is revealed in green and total DNA in red. Replication extent is 7% (Early, time after G1 release = 60 min), 51% (Mid, time = 90 min) and 98% (Late, time = 105 min).

Figure 4.

Saccharomyces cerevisiae temporal profile of normalized rate of origin firing and normalized fork density. I(t) and N_f(t) were normalized by their respective maximal values. (A) Saccharomyces cerevisiae temporal profile of normalized rate of origin firing from different experimental data sets [inverted triangle, DNA combing data from this work; open circle, microarray data from Yabuki et al. (8); open triangle, microarray data from McCune et al. (10); open square, microarray data from Raghuraman et al. (7)]. (B) Saccharomyces cerevisiae temporal profile of normalized replication fork density. The symbols are the same as in panel (A). (C) open circle, Population-averaged normalized rate of origin activation extracted from DNA combing; filled circle, single-cell normalized rate of origin firing obtained by Wiener Fourier deconvolution as described in the text. The solid red line is the 5 points smoothed curve. (D) open circle, Population-averaged normalized fork density extracted from DNA combing; filled circle, single-cell normalized fork density obtained by Wiener Fourier deconvolution as described in the text. The solid red line is the five points smoothed curve. In all panels, the origin of time axis was set to the starting time of the S phase (time = t−t_start).

Figure 5.

Normalized rate of origin firing versus normalized fork density extracted from DNA combing data in cell populations (A) and in single cells (B). The open black squares (open square) and the open grey circles (open circle) represent the increasing and decreasing part of I(t), respectively. The solid black and dashed grey lines are the spline smoothed curves in panel A, and the five points smoothed curves in panel (B).

Figure 6.

Modelling wild-type (dashed grey) and clb5Δ (solid black) rate of origin firing and fork density. (A and B) Normalized rate of origin firing (A) and fork density (B) from wild-type (open circle) and clb5Δ (open square) strains, extracted from McCune et al. (10). All data sets were normalized to the maximal value obtained for the wild-type strain. (C) Plot of the normalized rate of origin firing versus normalized fork density in the wild-type and clb5Δ strains. The increasing and decreasing parts of I(t) are indicated by arrows. (D) Simulated normalized rate of origin activation as a function of time. The dashed grey line represents the unmodified model (wild-type [WT]) and the solid black line the modified model (clb5Δ).