Fig 1.
Loss of Asf1 function impairs RDR.
(A) Diagram of the t>ura4<ori locus, in which t refers to the telomere-proximal side (gray lines), ura4 refers to the wt gene (blue line), > and < refers to the polarity of the two RTS1-RFBs (orange bars) and ori refers to replication origins (opened black circle, the largest one being the most efficient origin). Green bar indicates the rng3 probe. The RDR assay consists of a polar Replication Fork Barrier (RFB), called RTS1, integrated at the ura4+ gene, 5Kb away from a strong replication origin at the centromere-proximal side. An inverted repeated RTS1 sequence is integrated at the telomere-proximal side of ura4+ to generate the t>ura4<ori locus. Due to the main replication direction, the barrier activity of the centromere-proximal RTS1-RFB is predominant over the activity of the telomere-proximal RFB. The RFB activity is mediated by the RTS1-bound protein Rtf1, the expression of which is regulated by the nmt41 promoter repressed in the presence of thiamine. Upon Rtf1 expression, > 90% of forks are blocked at the centromere-proximal RFB. The binding of the Rad51 recombinase and its Rad52 loader allow the blocked fork to be restarted to overcome the RFB. Faulty restart events occur in ~ 2–3% of cells/replication: Rad51 promotes newly replicated strands to switch template and to invade the opposite inverted RTS1 sequence. DNA synthesis is then initiated from the 3’ invading strand on a non-contiguous DNA template, resulting in a stable early JM, referred to as a D-loop intermediate. Upon arrival of the converging fork, a second template switch event results in the formation of a later JM, referred to as Holliday junction (HJ)-like intermediate whose resolution generates at least two recombination products: acentric and dicentric which levels are a marker of RDR completion. (B) Top panel: Chromosome analysis in indicated strains and conditions by PFGE and Southern-blot using a radiolabeled rng3 probe. Bottom panel: Quantification of acentric level normalized to chromosome III level. Values are means of at least 3 independent biological replicates ± standard error of the mean (SEM). Statistical analysis was performed using student’s t-test: * p<0.05, ** p<0.005, compared to wt. (C) Left panel: schematics of replication intermediates (RIs) observed by 2DGE, in RFB OFF and ON conditions. Right panels: representative 2DGE experiments in indicated strains and conditions. White arrows indicate JMs. Numbers indicate the efficiency of the RFB for each strain analyzed, calculated as the % of arrested fork signal relative to Y arc signal, ± standard deviation (SD) (D) Quantification of percentage of JMs levels of panel C relative to replication fork barrier intensity. Individual values are plotted ± the range. See S1 Fig for RDR analysis in additional chromatin factor mutants.
Fig 2.
The mutated histone H3-H113D destabilizes (H3-H4)2 tetramer.
(A) Alignment of yeast, worm, mouse and human histone H3. Red rectangle indicates the position of histidine 113. UniProtKB access codes used are S. pombe: P09988; S. cerevisiae: P61830; C.elegans: P08898; M. musculus: P84228 and H. sapiens: Q71DI3. (B) View of the H3:H3’ interface. The interface between the two histones H3, colored here in light (H3) and dark blue (H3’), maintains H3 and H4 in the tetramer form (H3-H4)2. In the H3:H3’ interface, H113 of each histone (orange for H113 in H3 and red for H113 in H3’) is deeply buried in the adjacent histone partner. For clarity, the other histones H2A and H2B, as well as DNA, are not represented. (C) Left panel: schematic of the strains crossed. Right panel: spore viability analysis of indicated genotypes. (D) Left panel: association of H3-FLAG with H3-HA and H3-H113D-HA in indicated strains. Right panel: quantification expressed in arbitrary unit (a.u.). Individual values are plotted ± the range. (E) Left panel: association of H3-HA and H3-H113D-HA with untagged H3 and H4 in indicated strains. Right panels: quantification expressed in arbitrary unit (a.u.). Individual values are plotted ± the range. (F) Chromatin association of analyzed proteins in indicated strains and conditions (hours upon MMS addition or not). Two independent biological replicates were performed and representative blots are shown. See S2 Fig for structural impact of H3-H113D.
Fig 3.
H3-H113D alters replication-coupled chromatin assembly.
(A) Nucleosomal organization in indicated strains by analysis of MNase-digestion (0, 1, 3, 7.5 and 15 units) patterns of BrdU-incorporated genomic DNA. Total DNA (top) and corresponding BrdU-labelled replicated DNA (bottom) are shown. Densitometric profiles of the 7.5U MNase digestion product (arrows) of indicated strains are shown (left panels). Positions of the oligonucleosomes are indicated (mono and di nucleosomes). (B) Percentage of mono and di-nucleosome for the global chromatin (relative to total EtBr signal, left panel) and BrdU-positive mono and di-nucleosome (relative to total BrdU signal, right panel) according to the MNase units. Values are means of at least 3 independent biological replicates ± SEM. Statistical analysis was performed using student’s t-test: * p<0.05; **p<0.005, compared to wt. The yellow star on the left panel corresponds to the p value between wt and pcf1-d hht2-H113D. (C) Association of H3-HA and H3-H113D-HA with Pcf1-YFP and Asf1-MYC in indicated strains. See S3 Fig for BrdU incorporation and S4 Fig for protein-protein interactions.
Fig 4.
Histone deposition promotes TS during the initiation of RDR.
(A) Chromosome analysis in indicated strains and conditions as described on Fig 1. (B) Quantification of acentric level as described on Fig 1. Values are means of at least 3 independent biological replicates ±SEM. Statistical analysis was performed using student’s t-test: * p<0.05, *** p<0.0005, compared to wt. (C) Representative 2DGE analysis in indicated strains. White arrows indicate JMs. Numbers indicate the efficiency of the RFB for each strain analyzed, calculated as the % of arrested fork signal relative to Y arc signal ± SD (D) Quantification of percentage of joint-molecule levels of panel C relative to replication fork barrier intensity. Values are means of at least 3 independent biological replicates ±SEM. Statistical analysis was performed using student’s t-test: * p<0.05, ** p<0.005, compared to wt. (E) Rad52 binding to the t>ura4<ori locus in RFB OFF and ON conditions. Schematic at the bottom depicts the primers location within the t>ura4<ori locus: primer pairs 1 and 2 are located 400 and 200 bp away from the telomere-proximal RFB, respectively; primer pair 3 is within the ura4 gene; primer pairs 4, 5 and 6 are located 110, 450, and 630 bp away from the centromere-proximal RFB. Values are means of 4 independent biological replicates ±SEM. Statistical analysis was performed using Mann and Whitney U test: * p<0.05. See S5 Fig for the role of H3K56Ac in RDR.
Fig 5.
CAF-1 association to HR-mediated DNA synthesis requires Rad52.
Pcf1-YFP binding to the t>ura4<ori locus in RFB OFF and ON conditions (left panel: wt strain, middle panel: rad52-d strain, right panel: hht2-H113D strain). Schematic at the bottom depicts the primers location within the t>ura4<ori locus: primer pair 2 is located 200 bp away from the telomere-proximal RFB; primer pair 3 is within the ura4 gene; primer pair 4 is located 110 bp away from the centromere-proximal RFB. Values are means of at least 4 independent biological replicates ±SEM. Statistical analysis was performed using student t-test: * p<0.05, ** p<0.005, *** p<0.0005 compared to RFB OFF condition.
Fig 6.
Histone deposition ensures cell survival to camptothecin in the absence of Rqh1.
(A) Ten-fold serial dilution of indicated strains in indicated conditions of CPT and MMS. (B) Cell survival curves of indicated strains in indicated conditions. Values are means of at least 3 independent biological replicate ±SEM. Statistical analysis was performed using student t-test: * p<0.05, *** p<0.0005. (C) Ten-fold serial dilution of indicated strains in indicated conditions of bleomycin (BLEO).
Fig 7.
Histone deposition favors deletion-type events.
(A) Schematic representation of HR substrate and recombination outcomes. (B) Top panel: rate of conversion and deletion type in indicated strains. Values are median rate calculated from 23 to 26 independent cultures ± 95% confidence interval (CI). Bottom panel: ratio of deletion and conversion type in indicated strains. Error bars indicate SEM. Statistical analysis was performed using Mann & Whitney U test. Black and red stars indicate statistics compared to wt and compared to rqh1-d, respectively. * p<0.01, ** p<0.001.
Fig 8.
Model of histone deposition coupled to HR-mediated DNA synthesis at arrested forks.
Model of histone deposition coupled to recombination-dependent replication. Upon fork arrest, HR factors promote D-loop formation which primes the restarted DNA synthesis and histone deposition. Histone deposition coupled to RDR allows JMs to be protected from disassembly by DNA helicases such as Rqh1. This fork-associated histone deposition pathway to stabilize recombination intermediates contributes to replication recovery and balances genome stability at site of replication stress.