Histone deposition promotes recombination-dependent replication at arrested forks

RDR-coupled histone deposition promotes cell resistance to camptothecin in the absence of Rqh1

We have addressed the role of fork-associated histone deposition to the cell response to replication stress. The H3-H113D mutation or the pcf1 deletion decreased cell survival of rqh1 deleted cells upon camptothecin (CPT, a topoisomerase I inhibitor) treatment, and resulted in a moderate synthetic growth defect upon MMS treatment (Fig 6A and 6B). This was not a consequence of a loss of Rqh1-Pcf1 interaction in cells expressing H3-H113D as Rqh1-MYC associated with Pcf1-YFP in hht2-H113D mutated cells (S4D Fig). Also, DNA damage did not stimulate chromatin-bound H3-H113D-HA, suggesting that unstable tetramers are unlikely to be assembled during repair synthesis (Fig 2F). Interestingly, such synthetic interactions were not observed in response to bleomycin (Fig 6C), a DSB-inducing agent. These data reveal that in the absence of Rqh1 activity, cell resistance to topoisomerase I inhibitor relies on CAF-1-mediated histone deposition.

(H3-H4)₂ tetramer formation favors deletion type recombinant in the absence of Rqh1

We asked if the role of fork-associated histone deposition in promoting HR event might also occur independently of Rqh1. The lack of Rqh1 results in a spontaneous hyper-recombination phenotype caused by its multiple roles in processing HR intermediates [50]. We asked if histone deposition contributes to the anti-recombinase activity of Rqh1 by monitoring spontaneous HR events using an assay for intra-allelic recombination between direct ade6 repeats (Fig 7A) [51]. Gene conversion (GC) and synthesis dependent strand annealing (SDSA) result in conversion type, whereas crossover (CO) and single strand annealing (SSA, a DNA repair pathway independent of Rad51-mediated strand exchange) give rise to deletion type. In rqh1-d cells, conversion and deletion rate were increased by 2- and 3-fold, respectively, which is consistent with known Rqh1 anti-recombinase activities (Fig 7B) [50]. The hht2-H113D mutation had minor impact on HR outcomes but when combined with rqh1-d, it decreased by 35% the rate of deletion type without affecting the rate of conversion type (hht2-H113D and rqh1-d hht2-H113D compared to wt and rqh1-d respectively). These data establish that a significant part of the deletion events occurring in the absence of Rqh1 are caused by (H3-H4)₂ tetramer formation. Thus, histone deposition also promotes HR events by other mechanisms than counteracting Rqh1 activity. These data are consistent with antagonistic activities of Rqh1 and histone deposition in controlling HR outcomes: Rqh1 promotes D-loop disassembly to favor a conservative pathway whereas histone deposition promotes D-loop stability to favor deletion events such as CO.

Discussion

Fork-obstacles open up the risk of genome instability. HR ensures the timely completion of genome duplication by restarting dysfunctional forks, but this comes at the expense of TS events that can generate genome rearrangements. Here, we established that HR-mediated DNA synthesis is coupled to histone deposition, mediated by CAF-1 and Asf1, and that this process is necessary to promote RDR (Fig 8). We establish that inhibiting (H3-H4)₂ tetramer formation impacts RDR and D-loop resolution, in a manner dependent on CAF-1 function. Our data support a mechanism during which RDR is coupled to CAF-1-mediated histone deposition to stabilize D-loop intermediates to promote RDR. We reveal a novel interplay between chromatin restoration and DNA repair factors; a crosstalk necessary to cell survival to replication stress. Our finding highlight that chromatin restoration is an integral part of the RDR process. We proposed that histone deposition plays an active role in RDR to avoid discontinuity in chromatin assembly upon replication stress, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability.

A complex network of histone chaperones handles histone dynamics at replication forks to promote chromatin assembly. We establish that only a subset of this network is pivotal to promote RDR. Despite recent advances in understating the mechanism of CAF-1-mediated histone deposition in vitro, it remains challenging to define point mutations to generate mutated forms of CAF-1 unable to interact with H3-H4 in vivo [12]. To overcome this, we took advantage of the H3-H113D mutated form, reported to inhibit CAF-1-mediated histone deposition in vitro [36]. We show that H3-H113D precludes (H3-H4)₂ tetramer formation, is poorly incorporated into nucleosomes assembled onto DNA and alters the organization of the newly replicated chromatin. Remarkably, the H3-H113D mutation mimics the absence of CAF-1 in impairing RDR. We cannot exclude that H3-H113D has potential CAF-1-independent effects, but we have observed no additive effect between the lack of CAF-1 and H3-H113D indicating that the inhibition of replication-coupled histone deposition is responsible for the defect in RDR. RDR requires Asf1, the 3 CAF-1 subunits and the ability of CAF-1 to interact with PCNA [34], supporting the view that Asf1 and CAF-1 promote RDR by coupling histone deposition to the step of DNA synthesis. Consistent with this, CAF-1 binds to site of HR-mediated DNA synthesis in a Rad52-dependent manner, indicating that the early steps of RDR must be engaged for the subsequent recruitment of CAF-1. The binding of Rad52 to arrested forks is not affected when inhibiting histone deposition or in the absence of CAF-1 [34], consistent with CAF-1-mediated histone deposition acting downstream of Rad52.

We proposed that CAF-1-mediated histone deposition acts as a chromatin restoration pathway during the DNA synthesis step of RDR. In this scenario, histone deposition would occur onto the DNA duplex of the extended D-loop (Fig 8). We cannot exclude that histone deposition occurs onto the displaced strand of the D-loop, as it has been proposed that nucleosome can be deposited onto ssDNA [52]. RDR requires two histone chaperones that mediate chromatin assembly in a DNA synthesis manner and requires the ability of CAF-1 to interact with PCNA. Therefore, we favor the first hypothesis in which histone deposition is coupled to DNA synthesis during D-loop extension. Extensive works have address the role of chromatin factors in regulating genome accessibility to DNA repair machineries but how chromatin restoration is coupled to the DNA repair event is poorly understood [3]. Our data put forward a crosstalk between the DNA repair machinery and the step of chromatin restoration that plays an active role in promoting RDR.

Histone deposition coupled to RDR impacts D-loop resolution. One mechanism involves nucleosome assembly onto D-loop intermediates to counteract Rqh1 activity. Possibly, histone deposition onto extended D-loops creates a substrate less favorable to the Rqh1 activity. RecQ helicases act as motor proteins/helicases to migrate DNA junctions which may be prevented by assembled nucleosomes. It remains challenging to address whether histones are deposited onto D-loops as these structures are very transient. JMs visualization by 2DGE requires a step of enrichment in replication intermediates, technically incompatible with chromatin immuno-precipitation approaches to address histone binding to JMs. Nonetheless, we establish that inhibiting replication-coupled histone deposition favors D-loop disassembly. We provide insights into the nature of the chromatin required to protect D-loops. Asf1 acts as an histone chaperone to present H3-H4 to Rtt109 and generates the H3K56Ac mark and then transfer H3-H4 to CAF-1 [7]. We found H3K56Ac being dispensable to promote TS during the initiation of RDR. Possibly, Asf1 acts as a donor histone to CAF-1. However, H3-H113D binds to CAF-1 likely as a H3-H4 dimer but not to Asf1, indicating that H3-H4 can be handed off to CAF-1 independently of Asf1, a pathway that remains to be clarified.

Nucleosome deposition onto extended D-loop may impose topological constraints. D-loop disassembly is a topoisomerase-mediated mechanism [53]. Thus, topological constraints resulted from DNA wrapped around nucleosome may be easily relieved by topoisomerase 3. When seeking for additional chromatin factors required for RDR, we found Nap1 and Nap2, two histone H2A-H2B chaperones, to be dispensable to protect D-loop. This suggests that the deposition of (H3-H4)₂ tetramer, but not the formation of a nucleosome, is sufficient by itself to counteract Rqh1 activity and to limit topological constraints.

Fork-associated histone deposition affects HR outcomes by mechanisms that are also independent of Rqh1 activity. During HR-mediated DSB repair, disassembly of D-loops extended by DNA polymerase ensures a non-crossover outcome [26]. We report that in the absence of Rqh1, a significant part of the deletion type events is favored by histone deposition. Chromatin assembly at D-loop intermediates may also counteract the activity of other helicases in disrupting JMs [26]. Interestingly, spontaneous rates of gene conversion were unaffected by inhibiting histone deposition. This suggests that DNA synthesis associated to GC and SDSA is too short in length to favor histone deposition. Nonetheless, our data reveal that the antagonistic activities of RDR-coupled histone deposition and Rqh1 in D-loop resolution are pivotal to balance genome stability at arrested forks and to promote cell resistance upon replication stress.

During unchallenged replication, the concerted action of multiple histone chaperones coordinates the assembly of chromatin behind the fork to achieve recycling of parental histones and deposition of newly synthetized histones [20]. RDR results in the progression of non-canonical forks in which both strands are synthetized by the DNA polymerase delta, which contrasts with the division of labor between DNA polymerase delta and epsilon at origin-born replication forks [54]. Such restarted forks are liable to replication errors such as multiple template switches, replication slippage and U-turn [32,33,55]. Despite these unusual features, our data suggest that restarted forks remain coupled to histone deposition and thus may help to ensure continuous chromatin assembly upon replication stress.

Fork obstacle and replication stress interfere with the inheritance of epigenetic marks [23,56,57]. On the one hand, the post-replicative repair of DNA lesions/structures, left un-replicated behind the fork, is uncoupled from chromatin assembly and recycling of parental histones. On the other hand, it was proposed that the bypass of DNA secondary structures, such as G quadruplexes, by PrimPol allows the repriming of DNA synthesis very closely to the fork obstacle, allowing for the maintenance of replication-coupled chromatin maturation [58]. The choice of the pathway employed to overcome fork obstacles may impact on the maintenance of histone deposition coupled to restarted forks. Our data indicate that RDR, a main pathway to bypass fork obstacles, is coupled to histone deposition. In the view that replisomes are often interrupted by numerous obstacles, RDR-coupled histone deposition contributes to cell resistance to replication stress and may ensure continuity in assembling chromatin upon replication stress. Nonetheless, this comes at the expense of stabilizing JMs which can be detrimental to genome stability.

Material and methods

Standards yeast genetics

Yeast strains used in this work are listed in S1 Table. Gene deletion and gene tagging were performed by classical and molecular genetics techniques [59]. Strains containing the RTS1-RFB were grown in supplemented EMM-glutamate media containing 60μM of thiamine. To induce the RTS1-RFB, cells were washed twice in water and grown in supplemented EMM-glutamate media containing thiamine (Rtf1 repressed, RFB OFF condition) or not (Rtf1 expressed, RFB ON condition) for 24 hours or 48 hours.

Chromatin fraction assay

Chromatin fractionation was performed as described previously [60] with the following modifications: Cells were harvested, digested with 100μg of zymolyase 20T (Amsbio, 120491–1) to obtain spheroplasts, and resuspended in lysis buffer (50mM potassium acetate, 2mM MgCl2, 20mM HEPES pH7.9, 1% Triton X-100, 1mM PMSF, 60mM β-glycerophosphate, 0.2mM Na₃VO₄, 1μg/ml AEBSF and Complete Protease Inhibitor EDTA-Free Tablet (Roche, 04 693 159 001). After lysis, extracts were subsequently fractionated into soluble and pellet fractions by centrifugation. The insoluble chromatin-enriched pellet fraction was washed twice with the lysis buffer without 1% Triton X-100 and digested with 100Units of DNase I HC (ThermoScientific, EN0523) on ice for 15min followed by 15min at RT. The DNase I-digested chromatin-enriched fraction was centrifuged for 5 min at 16,000g. Supernatant was designated as the chromatin fraction. Samples corresponding to total soluble and chromatin fractions were migrated and transferred on nitrocellulose membrane. Proteins of interest were revealed by anti-HA (1/500^e, SantaCruz, 12CA5), anti-PCNA (1/500^e, SantaCruz, PC10), anti-Tubulin (1/4000^e, Abcam, ab6160) and anti-H3 (1/2000^e, Abcam, ab1791) antibodies.

Analysis of replication intermediates by 2DGE

Replication Intermediates (RIs) were analyzed by 2DGE as previously described [61]. RIs were migrated in 0.35% agarose gel in 1X TBE for the first dimension. The second dimension was migrated in 0.9% agarose gel 1X TBE supplemented with EtBr (Brewer 1992). DNA was transferred onto a nylon membrane (Perkin Elmer, NEF988001PK) in 10X SSC. Membranes were incubated with a ³²P radiolabeled ura4 probe, and RIs were detected using phosphor-imager software (Typhoon-trio) and quantified with ImageQuantTL.

Co-immunoprecipitation

5.10⁸ cells were harvested with 0.1% sodium azide, washed in cold water and resuspended in 400 μl of EB buffer (50mM HEPES High salt, 50mM KOAc pH7.5, 5mM EGTA, 1% Triton X-100, 1mM PMSF, and Complete Protease Inhibitor EDTA-Free Tablet (Roche, 04 693 159 001). Cell lysis was performed with a Precellys24 homogenizer (Bertin instruments). The lysate was treated with 250mU/μl of benzonase (Novagen, NOVG 70664–3) for 30min. After centrifugation, the supernatant was recovered and an aliquot of 50 μl was saved as the INPUT control. To 300μl of lysate, 2μl for anti-GFP (Life Technologies, A11122) were added and incubated for 1.5 hours at 4°C on a wheel. Then, 20μl of Dynabeads protein-G (Life Technologies, 10004D) prewashed in PBS were added and then incubated at 4°C overnight. Alternatively, lysates were incubated overnight with 20μl of anti-MYC (Life Technologies, 88842) or anti-HA (Life Technologies, 88836) antibody coupled to magnetic beads. Proteins of interest were detected using anti-HA high affinity (1/500e, Roche, clone 3F10), anti-GFP (1/1000e Roche, 11 814 460 001), anti-MYC (1/300e, SantaCruz, A-14), anti-HA (1/500e, SantaCruz, 12CA5), anti-PCNA (1/500e, SantaCruz, PC10), and anti-H3 (1/2000e, Abcam, ab1791) antibodies. Co-immunoprecipitation experiments were quantified with ImageLab software (BioRad). Intensities of bands were normalized with the background.

Chromatin immunoprecipitation of Pcf1-YFP

ChIP experiments were performed as previously reported [62]. 10⁹ Cells were crosslinked with fresh 1% formaldehyde (Sigma, F-8775) for 15 minutes. Cells were lysed using Precellys24 homogenizer (Bertin instruments) in lysis buffer (50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1mM phenylmethylsulfonyl fluoride, and Protease Inhibitor Cocktail (Sigma P8215)). The crude cell lysate was sonicated (using a Diagenod Bioruptor at high setting for 15 cycles: 30 seconds ON + 30 seconds OFF) and clarified by centrifugation for 15min at 16,000g. Prior to immunoprecipitation, 1/100 volume of the cell lysate was saved for an input control. Immunoprecipitations were performed with 2μl of anti-GFP antibody (Life Technologies, A11122) for 2 hours. After 30min incubation with 20μl magnetic beads (Life Technologies, 10004D), immunoprecipitates were successively washed with 2x1ml lysis buffer, 2x1ml lysis buffer/500mM NaCl, 2x1ml wash buffer (10mM Tris-HCl pH8, 0.25M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1mM EDTA) and 1ml TE buffer (10mM Tris-HCl, 1mM EDTA pH8). Crosslink was reversed by incubating the samples at 65°C overnight. Samples were then treated with 0.5mg/ml Proteinase K (Euromedex, EU0090) and DNA was purified using Qiagen PCR purification kit and eluted in 100μl of water. The relative amount of DNA was quantified by qPCR (primers are listed in S2 Table). Pcf1-YFP enrichment was normalized to an internal control locus (ade6).

Chromatin immunoprecipitation of Rad52

Rad52 ChIP experiments were performed as previously reported [63]. 2.10⁸ cells were fixed with 1% formaldehyde (Sigma, F-8775), lysed in 500μl breaking buffer (0.1M Tris-HCl pH8, 20% glycerol, 1mM phenylmethylsulfonyl fluoride) using a Precellys24 homogenizer (Bertin instruments) and sonicated with a Diagenod Bioruptor at high setting for 15 cycles: 30 seconds ON + 30 seconds OFF). Immunoprecipitations (IP) were carried out overnight at 4°C in ChIP buffer (50mM Hepes-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1mM phenylmethylsulfonyl fluoride) using 1μl of anti-Rad52 (Abcam, Ab63800). Protein G magnetic beads (Life Technologies, 10004D) were then added to the samples and incubated for 4h at 4°C. IPs were successively washed with 1ml ChIP buffer, 2x1ml ChIP buffer/500mM NaCl, 2x1ml wash buffer (10mM Tris-HCl pH8, 0.25M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1mM EDTA, 1mM phenylmethylsulfonyl fluoride) and 1ml TE buffer (10mM Tris-HCl, 1mM EDTA pH8). Crosslinking was reversed for both IP and Input samples by incubation at 65 °C overnight. Samples were then treated with 0.5mg/ml Proteinase K (Euromedex, EU0090) and DNA was purified by phenol/chloroform extraction. Purified DNA were analyzed by real-time quantitative PCR (CFX96 Real-Time PCR Detection System and iQ SYBR Green Supermix, BioRad) using those cycling conditions: 95°C for 3 min; 40 cycles of 95°C for 15 seconds, 60°C for 1 min. Primers used in this study are listed in S2 Table.

Pulse-Field Gel Electrophoresis

PFGE were performed as previously described [27]. Membranes were then incubated with a ³²P radiolabeled rng3 probe. Quantification of acentric chromosomes visualized by PFGE was performed as previously described [34].

Microccocal digestion and BrdU incorporation

Microccocal digestions were performed as previously described [64]. After crosslink with 1% formaldehyde, 1.10⁹ Cells were spheroplasted in 2ml of CES buffer (50mM Citric acid/50mM Na2HPO₄ pH 5.6, 40mM EDTA pH8, 1.2M Sorbitol, 20mM β-mercaptoethanol) containing 1 mg/ml Zymolyase 100T (Amsbio, 120493–1) for 20min at 30°C. Spheroplasts were washed twice with 1ml of iced cold 1.2M Sorbitol buffer. Cells were resuspended in 1ml of NP-S buffer (1.2M Sorbitol; 10mM CaCl₂, 100mM NaCl, 1mM EDTA pH8, 14mM β-mercaptoethanol, 50mM Tris-HCl pH8, 0.075% NP-40, 5mM spermidine, 0.1mM PMSF, Complete Protease Inhibitor EDTA-Free Tablet (Roche, 04 693 159 001)) containing the indicated units of Micrococcal Nuclease (Worthington Biochemical, LS004798) for 10 min at 37°C. Reactions were stopped by addition of 50mM EDTA pH8 and SDS 0,2%. Crosslinking was reversed overnight at 65°C in the presence of 20μg of RNAseA (Sigma, R5503) and 0.2mg/ml Proteinase K (Euromedex, EU0090). DNA was purified by phenol/chloroform extraction and ethanol precipitation. Purified DNA was resolved on 1.5% agarose gel (1X TBE).

For BrdU incorporation, we used cells expressing Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) under the control of the fission yeast adh promoter, together with the human equilibrative nucleoside transporter (hENT1) (adh-dmdNK-adh-hENT1) [44]. Cells were arrested 4 hours with 40mM HU (Sigma, H8627) and released in fresh media containing BrdU (Sigma, B5002) for 20min. After MNase digestion, DNA was analyzed by Southern-blot using anti BrdU antibody (1/4000^e, Abcam, ab12219).

Spontaneous recombination assay

Spontaneous recombination rate was assayed using strains containing a direct repeat of two nonfunctional ade6 alleles flanking a functional ura4 gene [51]. Strains were kept on low adenine EMM plates lacking uracil to prevent selection for Ade⁺ and Ura^- recombinants. Dark pink colonies were streaked on supplemented YE plates and 23 to 26 independent single colonies for each strain were used to calculate Ade+ recombinant rate. Appropriate dilutions were plated on supplemented YE plates (to determine cell survival), EMM plates lacking adenine (to score spontaneous recombination rate, Ade+ recombinants) and EMM plates lacking adenine and uracil (to score gene conversion rate, Ade+ Ura+ recombinants). Colonies were counted after 5–7 days of incubation at 30°C. The rates of Ade+ and Ade+ Ura+ recombinant were calculated using the as described in [65].

Analyzed X-ray structures of nucleosome

There is no experimental model of nucleosome containing histones from S. pombe. Among the numerous available structures of nucleosome, only three of them include yeast histones, all from S. cerevisiae (PDB codes 1ID3, 4JJN and 4KUD), with resolutions of ~3 Å. Indeed, the highest resolution was obtained for a nucleosome containing histones from Xenopus laevis (PDB code 1KX5, resolution of 1.9 Å). Since the histone sequences are extremely conserved, S. cerevisiae and Xenopus laevis H3 histones share 92% of residues with H3 of S. pombe. More specifically, the region surrounding H113 is well preserved across S. pombe, S. cerevisiae and Xenopus laevis, with a very good score being observed for the couple S. pombe / Xenopus laevis (S2A Fig). Given the reasonable sequence agreement, 1ID3, 4JJN, 4KUD and 1KX5 were analyzed using PDBsum to describe the interface between two H3-H4 dimers [66].

Data availability

Numerical data used for graphs are provided in the supplementary information S1 Data.

Supporting information

S1 Fig [a]
Chromatin factors involved in RDR (related to ).

S2 Fig [a]
Impact of H3-H113D on interactions with H3 (related to ).

S3 Fig [tif]
BrdU incorporation during and after HU treatment (related to ).

S4 Fig [a]
Impact of H3-H113D on interactions with histone chaperones (related to ).

S5 Fig [a]
H3K56Ac is dispensable to promote TS during the initiation of RDR (related to ).

S1 Table [docx]
Strains used in this study (related to all figures).

S2 Table [docx]
List of primers used in this study.

S1 Data [xlsx]
Excel file containing numerical raw data.