ATP-dependent chromatin remodeling shapes the DNA replication landscape

Abstract

The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 have especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes that promote DNA replication and define a specific stage of replication that requires remodeling for normal function.

Figure 1

MMS sensitivity of isw2 nhp10 mutants is due to a prolonged S phase. (a) isw2 nhp10 double mutants grow slowly in the presence of MMS. Wild type (W1588-4c), isw2 (YTT1080), nhp10 (YTT2060), isw2 nhp10 (YTT2109) strains were grown to saturation then 10-fold serial dilutions were plated onto YPD plates with or without 0.02% MMS. (b) S phase is prolonged in MMS-treated isw2 nhp10 mutants. The above strains were arrested in G1 and then released into S phase in the presence of 0.02% MMS as diagrammed. Cells were collected at the indicated times after release and DNA content was determined by flow cytometry (black lines). Gray profiles are from asynchronous cells collected prior to G1 arrest.

Figure 2

Isw2 and Ino80 complexes are required for efficient replication of late-replicating regions in the presence of MMS. (a–d) Replication profiles of chromosomes IV (a), VI (b), XV (c) and III (d) from WT (YTT1831) and isw2 nhp10 (YTT3306) strains undergoing S phase in the presence of MMS. (e) Close view of the replication profile at the interface between early and late-replicating regions on chromosome IV at chromosomal coordinates 550–700 kb (dotted box in a). Profiles were generated from cells collected at 30 (black), 45 (light blue), 60 (green), 90 (orange), 120 (dark blue), and 150 (grey) minutes after release from G1 arrest. Collection of isw2 nhp10 samples was initiated at 45 minutes due to a less efficient release from α-factor arrest characteristic of this strain (See Supplementary Fig. 2b, 4b). Positions of confirmed and likely ARSs are indicated at the bottom of each graph. Triangles correspond to positions of origins that are replicated early in a normal S phase (filled triangles represent origins that are amongst the first 25% replicated in two studies of replication timing,; open triangles represent origins that are amongst the earliest 25% in only one of the two studies). The filled circles correspond to remaining origins. Origins that are fired in a wild type strain during DNA damage checkpoint activation by 200mM Hydroxyurea (early firing origins) are indicated in red. The black circle on the x-axis indicates position of the centromere. The red and grey boxes represent approximations of early and late-replicating regions respectively.

Figure 3

Replication-fork progression is slowed in isw2 nhp10 mutants. (a) Diagram of the right arm of chromosome VI in the strains used for replication-fork progression measurements. The locations of the five regions (Regions 1–5) monitored for replication are shown in shades of blue. ARS608 and ARS609 (white boxes) are deleted to promote unidirectional replication from ARS607 to the telomere. (b) Replication kinetics along chromosome VI. Percent replication of Regions 1–5 (colors correspond to diagram in a) was determined throughout S phase in the presence of MMS. The dashed line corresponds to the percentage of budded cells. The horizontal dotted line corresponds to the percent replication value one-half of the maximum obtained for that experiment. The T_rep for that region is indicated on the x-axis by the arrowhead of the same color. Wild type (YTT3528) and isw2 nhp10 (YTT3531) strains were treated as in Supplementary Figure 2a. The kinetic curves of replication in the double mutant are more flat than those of wild type cells because the mutant is released from G1 arrest in a less synchronous fashion (see the budding index and Supplementary Fig. 4b). Importantly, the difference in the release kinetics only affects the slope of kinetic curve at each time point, not the distance between each line that reflects replication fork rate. (c) Replication times relative to Region 1. The values for T_rep determined in (b) were plotted relative to the value for Region 1 and are indicated by arrows (colors correspond to diagram in a). The white arrows indicate the time of half-maximal budding in the population (T_bud).

Figure 4

Isw2 and Ino80 may directly facilitate DNA replication. (a) Venn diagram showing the lack of overlap of genes deactivated 1.5 fold in isw2 nhp10 (gray) with genes involved in replication, repair, checkpoint (yellow), and MMS resistance (blue). The 93 genes represented are a collection of genes that are deactivated in isw2 nhp10 mutants in comparison to isw2 and nhp10 single mutants under normal or MMS growth conditions. The Venn diagram encompasses data from 6144 genes. (b) Chromatin immunoprecipitation (ChIP) of Pol1, Nhp10, and Isw2 during S phase. YTT3735 and YTT3736 cells (Pol1-3Flag, Nhp10-13Myc, Isw2-Avi) were arrested in G1 and released into S phase in the presence of 200 mM HU. Cells were harvested at indicated time points in S phase and utilized for ChIP analysis. DNA from immunoprecipitated fractions was analyzed by radioactive PCR with primers corresponding to an early origin (ARS607) and a late origin (ARS1502). ARS1502 does not initiate replication under this experimental condition. The mean and standard deviation of the signals at ARS607 relative to ARS1502 from three biological replicates are presented.

Figure 5

Isw2 and Ino80 promote replication in the absence of MMS. (a) A clb5 deletion causes a severe growth defect in the absence of Isw2 and Nhp10. Wild type (W1588-4c), isw2 (YTT1080), nhp10 (YTT2060), isw2 nhp10 (YTT2109), clb5 (YTT3402),isw2 clb5 (YTT3434) nhp10 clb5 (YTT3437), and isw2 nhp10 clb5(YTT3441) were grown on YPD media at 30°C for 2 days. (b) A mec1 deletion suppresses the extended S phase of isw2 nhp10 mutants in the presence of 0.02% MMS. mec1 (YTT3003) and mec1 isw2 nhp10 (YTT3048) mutants were treated and their DNA content determined as in Figure 1b. WT and isw2 nhp10 FACS profiles are reproduced from Fig.1b for comparison. Both strains contain an sml1 deletion to suppress lethality of a mec1 deletion.