Figure 1.
H2Bub regulates fork stalling in HU.
(A) The response of WT (CFK1204) and htb-K123R (CFK1231) cells to acute doses of HU. Log-phase cells were treated with 0.2M HU for the indicated times, and dilutions were subsequently spread onto YPD plates. The plates were incubated at 30°C for 2–3 days and viability was estimated based on colony forming units (CFU). Viability was normalized to 0 min of HU treatment, which was set as 100%. (B) Flow cytometry was used to analyze the cell cycle progression of WT (CFK1204) and htb-K123R (CFK1231) cells in the presence of 0.2M HU for 120 minutes after release from α-factor-induced G1 arrest. DNA content is visualized by propidium iodide incorporation. (C–E) Replication profiles of replication origins: (C) ARS305 and ARS310, (D) ARS603, ARS606, and ARS607, and (E) ARS1309/1310 and ARS1312, in WT (CFK1419) and htb-K123R (CFK1421) cells. Cells were synchronized in G1 with α-factor, and then released into media containing 0.2M HU and 200 µg/ml BrdU for 90 minutes. After DNA extraction and fragmentation, BrdU-labeled DNA was immunoprecipitated and hybridized on high-resolution oligonucleotide tiling arrays. Orange histogram bars (BrdU) on the y axis represent the average signal ratio on a log2 scale of loci along the reported regions. Positions of ARS elements are identified by Mcm2 occupancy [72]. (F) The graph depicts the distribution of BrdU track lengths in WT (CFK1419) and htb-K123R (CFK1421) cells. Box and whiskers indicate the minimum, maximum, and 25–75 percentiles, respectively. Mean BrdU tracks lengths are indicated in kb. Asterisks indicate the P-value of the statistical test (Mann–Whitney rank sum t-test, ** P-value<0.005).
Figure 2.
H2Bub-mediated fork stalling is independent of dNTP pool size.
The size of the dNTP pools in exponentially-growing cultures of WT (CFK1419), htb-K123R (CFK1421), dun1Δ (YCL023), and dun1Δ htb-K123R (YCL025) cells in YPD media. Two independent isogenic strains of each genotype were analyzed. (B) Graph depicting the distribution of BrdU track lengths in WT (CFK1419), htb-K123R (CFK1421), dun1Δ (YCL023), and dun1Δ htb-K123R (YCL025) mutants, as shown in Fig. 1F. (C) Replication profiles of the replication origins ARS207, ARS208/209, ARS718, ARS719/720, and ARS1309/1310 in WT (CFK1419), htb-K123R (CFK1421), dun1Δ (YCL023), and dun1Δ htb-K123R (YCL025) mutants. The BrdU histogram was analyzed as described in Fig. 1C–E. (D) Temperature sensitivity and HU resistance of the indicated genotypes (WT (CFK1204), sml1Δ (CFK1481), dun1Δ (YMW069), htb-K123R (CFK1231), and htb-K123R in combination with sml1Δ (CFK1482) or dun1Δ (YMW072)). Log-phase cells were serially diluted and spotted onto YPD plates with or without HU, and incubated at 30°C or 37°C for 2–3 days.
Figure 3.
H2Bub preserves replication fork stability under HU stress.
(A) Analysis of replication intermediates (RIs) at ARS305 and ARS607 in WT (CFK1204) and htb-K123R (CFK1231) mutants. Cells were synchronized at G1 phase and released into media containing 200 mM HU for 120 minutes. DNA was prepared from cells collected at the indicated times, cut with HindIII (ARS305) or SacI and ApaL1 (ARS607), and analyzed by 2D gel using the ARS305 or ARS607 probe, as described in the Materials and Methods. (B) Accumulation of damaged DNA in H2Bub-depleted cells. WT (CFK1204) and htb-K123R (CFK1231) cells were arrested in G1 and released into fresh media containing 0.2M HU for 90 minutes at 30°C. Whole cell lysates were prepared at the indicated time points, and analyzed by Western blot using antibodies against γ-H2A, a marker of DNA damage. G6PDH was used as a loading control. Asy: Asynchronized cells. (C) Cells lacking H2Bub are more sensitive to replication stress. Ten-fold serial dilutions of yeast cells (WT (CFK1204) and htb-K123R (CFK1231)) were spotted onto nonselective YPD plates under different temperatures or YPD containing 100 or 150 mM HU for a period of several days.
Figure 4.
The Bre1-H2Bub pathway genetically interacts with components of the intra-S-phase checkpoint.
(A) H2Bub functions in parallel with DNA polymerase (pol2-11) and intra-S-phase checkpoint cascades (Mec1, Sgs1, and Mrc1). WT and pol2-11 cells carrying HTB1 or the htb1-K123R allele on a HIS3 vector were transformed with HTB1 on a URA3 vector. The strains containing both URA3 and HIS3 (CFK2000, CFK2002, CFK2004, and CFK2006) were streaked onto 5-FOA plates to select for cells lacking H2Bub (htb1-K123R). Ten-fold serial dilutions of the indicated strains were spotted onto YPD plates in the absence or presence of different doses of HU at 30°C (WT (CFK1204, CFK2352, and CFK2414), htb-K123R (CFK1231 and CFK2416), mec1-100 (CFK2346), sgs1Δ (CFK1447), mrc1Δ (CFK1444), rad53-11 (CFK2347), and double mutants (CFK2356, CFK1453, CFK1450, and CFK2358)). (B) The H2B ubiquitin E3 ligase, Bre1, functions in parallel with intra-S-phase checkpoints under HU stress. Ten-fold serial dilutions of the indicated strains (WT (CFK2351), bre1Δ (YMW093), mec1-100 (CFK2346), mec1-100 bre1Δ (YMW095), sgs1Δ (CFK2371), bre1Δ sgs1Δ (CFK2373), rad53-11 (CFK2347), and rad53-11 bre1Δ (CFK2378)) were spotted onto YPD plates with and without HU as described in (A). (C) The response of double mutants of htb-K123R and mec1-100 or rad53-11 to acute exposure to HU. Logarithmically-growing cells were treated with 0.2M HU as described in Fig. 1A.
Figure 5.
H2Bub and Sgs1 play interdependent roles in Rad53 phosphorylation.
(A) Sgs1 occupancy at replication origins is unstable in htb-K123R cells exposed to HU. WT (CFK1764) or htb-K123R (CFK1765) cells were synchronized in G1 and then released into fresh YPD containing 0.2M HU for 60 minutes at 30°C. Chromatin immunoprecipitation (ChIP) was performed using antibodies against Sgs1-3×Myc. DNA was quantified by qPCR using primers adjacent to ARS305 and a region 3.5 kb distal. Sgs1 occupancy at each time point was normalized to that of G1. (B) Activation of Rad53 is impaired in the absence of both H2Bub and Sgs1. WT (CFK1204), htb-K123R (CFK1231), sgs1Δ (CFK1447), and sgs1Δ htb-K123R (CFK1453) cells were arrested in G1 and released into fresh media containing 0.2M HU for 90 minutes at 30°C. Whole cell lysates were prepared at the indicated time points, and analyzed by Western blot using antibodies against Rad53 (EL7), phospho-Rad53 (F9), H2B, and mono-ubiquitylated H2B (anti-FLAG). G6PDH was used as a loading control.
Figure 6.
H2Bub and Sgs1 cooperatively control replication fork stalling and stability under HU.
(A) Replication profiles of the early origins ARS305 and ARS607 in sgs1Δ (YCL007) and sgs1Δ htb-K123R (YCL008) mutants. The BrdU histogram was analyzed as described in Fig. 1C–E. (B) Graph depicting the distribution of BrdU track lengths in WT (CFK1419), sgs1Δ (YCL007), and sgs1Δ htb-K123R (YCL008) mutants, as shown in Fig. 1F. (C) Cell cycle progression of these mutants in the presence of 0.2M HU was analyzed by flow cytometry. (D) The size of the dNTP pools in exponentially-growing cultures of WT (CFK1419), htb-K123R (CFK1421), sgs1Δ (YCL007), and sgs1Δ htb-K123R (YCL008) cells in YPD media. Two independent isogenic strains of each genotype were analyzed. (E) Survival of WT (CFK1204), htb-K123R (CFK1231), sgs1Δ (CFK1447), and sgs1Δ htb-K123R (CFK1453) cells in response to acute doses of HU, as shown in Fig. 1A.
Figure 7.
H2Bub promotes chromatin assembly in response to replication stress.
(A) H2Bub is required for nucleosome assembly near replication forks under replication stress. WT (CFK1204) or htb-K123R (CFK1231) cells were arrested in G1 phase using α-factor, and were then released into 200 mM HU at 30°C for 60 minutes. At the indicated time, cells were collected and histone occupancy at two early origins (ARS305 and ARS607) and one late origin (ARS501) was determined by ChIP using antibodies against H3. IP signals at ARS sequences were normalized to IP signals at TELVI-R. The results are the mean +/− SEM of three replicates. (B) Genetic interactions between H2Bub and histone chaperones (Cac1, Asf1, and Hir1) or a histone acetyl-transferase (Rtt109). Ten-fold serial dilutions of the indicated strains (WT (CFK1204), htb-K123R (CFK1231), cac1Δ (CFK1206), cac1Δ htb-K123R (CFK1237), asf1Δ (CFK1208), asf1Δ htb-K123R (CFK1233), rtt109Δ (CFK1212), rtt109Δ htb-K123R (CFK1241), hir1Δ (CFK1202), and hir1Δ htb-K123R (CFK1235)) were spotted onto YPD plates containing HU (0 or 50 mM), and cell growth was monitored for 2–3 days. (C) The survival of asf1Δ (CFK1208) and asf1Δ htb-K123R (CFK1233) cells in response to acute treatment with HU, as described in Fig. 1A. (D) H2Bub modulates the interaction between Asf1 and Rad53 under HU stress. Asynchronous cultures of WT (YMW105) or htb-K123R (YMW104) cells were untreated (−) or treated (+) with 0.2M HU for 90 minutes. Protein extracts were prepared and incubated with pre-bound anti-HA-protein G beads to pull down Asf1-3×HA, and the immune-precipitates were resolved by SDS-PAGE, before being probed with either anti-HA or anti-Rad53 antibodies. (E) A working model depicting the role of H2Bub in nucleosome assembly under HU stress. H2Bub coordinates nucleosome assembly in response to replication stress by directly contributing to nucleosome formation and by indirectly regulating the availability of Asf1 during HU stress.
Figure 8.
A model for how H2B mono-ubiquitylation facilitates fork stability under replication stress.
Upon HU-induced stress, H2Bub promotes nucleosome assembly, which assists replication fork stalling, Sgs1 recruitment, and Rad53 phosphorylation. The reassembly of chromatin on nascent DNA restricts fork progression and promotes replication fork stability and its recovery after the removal of HU. In the absence of H2Bub (bre1Δ/htb-K123R), replication fork movement is faster than even that observed under nucleotide depletion by HU, which results in shorter tracts of RPA-coated-single-stranded DNA. This in turn reduces retention of Sgs1 at the forks, and delays phosphorylation of Rad53.