RNA:DNA hybrids from Okazaki fragments contribute to establish the Ku-mediated barrier to replication-fork degradation.

Nonhomologous end-joining (NHEJ) factors act in replication-fork protection, restart, and repair. Here, we identified a mechanism related to RNA:DNA hybrids to establish the NHEJ factor Ku-mediated barrier to nascent strand degradation in fission yeast. RNase H activities promote nascent strand degradation and replication restart, with a prominent role of RNase H2 in processing RNA:DNA hybrids to overcome the Ku barrier to nascent strand degradation. RNase H2 cooperates with the MRN-Ctp1 axis to sustain cell resistance to replication stress in a Ku-dependent manner. Mechanistically, the need of RNaseH2 in nascent strand degradation requires the primase activity that allows establishing the Ku barrier to Exo1, whereas impairing Okazaki fragment maturation reinforces the Ku barrier. Finally, replication stress induces Ku foci in a primase-dependent manner and favors Ku binding to RNA:DNA hybrids. We propose a function for the RNA:DNA hybrid originating from Okazaki fragments in controlling the Ku barrier specifying nuclease requirement to engage fork resection.

SUMO protease and proteasome recruitment at the nuclear periphery differently affect replication dynamics at arrested forks.

Nuclear pore complexes (NPCs) have emerged as genome organizers, defining a particular nuclear compartment enriched for SUMO protease and proteasome activities, and act as docking sites for the repair of DNA damage. In fission yeast, the anchorage of perturbed replication forks to NPCs is an integral part of the recombination-dependent replication restart mechanism (RDR) that resumes DNA synthesis at terminally dysfunctional forks. By mapping DNA polymerase usage, we report that SUMO protease Ulp1-associated NPCs ensure efficient initiation of restarted DNA synthesis, whereas proteasome-associated NPCs sustain the progression of restarted DNA polymerase. In contrast to Ulp1-dependent events, this last function is not alleviated by preventing SUMO chain formation. By analyzing the role of the nuclear basket, the nucleoplasmic extension of the NPC, we reveal that the activities of Ulp1 and the proteasome cannot compensate for each other and affect the dynamics of RDR in distinct ways. Our work probes two distinct mechanisms by which the NPC environment ensures optimal RDR, both controlled by different NPC components.

Unprotected Replication Forks Are Converted into Mitotic Sister Chromatid Bridges.

Replication stress and mitotic abnormalities are key features of cancer cells. Temporarily paused forks are stabilized by the intra-S phase checkpoint and protected by the association of Rad51, which prevents Mre11-dependent resection. However, if a fork becomes dysfunctional and cannot resume, this terminally arrested fork is rescued by a converging fork to avoid unreplicated parental DNA during mitosis. Alternatively, dysfunctional forks are restarted by homologous recombination. Using fission yeast, we report that Rad52 and the DNA binding activity of Rad51, but not its strand-exchange activity, act to protect terminally arrested forks from unrestrained Exo1-nucleolytic activity. In the absence of recombination proteins, large ssDNA gaps, up to 3 kb long, occur behind terminally arrested forks, preventing efficient fork merging and leading to mitotic sister chromatid bridging. Thus, Rad52 and Rad51 prevent temporarily and terminally arrested forks from degrading and, despite the availability of converging forks, converting to anaphase bridges causing aneuploidy and cell death.

Restarted replication forks are error-prone and cause CAG repeat expansions and contractions.

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2-7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

Histone deposition promotes recombination-dependent replication at arrested forks.

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability.

Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange.

Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

The chromatin assembly factor 1 promotes Rad51-dependent template switches at replication forks by counteracting D-loop disassembly by the RecQ-type helicase Rqh1.

At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop.

Recovery of arrested replication forks by homologous recombination is error-prone.

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

Disordered regions and folded modules in CAF-1 promote histone deposition in Schizosaccharomyces pombe.

Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.

A marker-free genome editing method in S. pombe using the delitto perfetto approach.

The fission yeast, like budding yeast, offer an easy manipulation of their genome, despite their distinct biology. Most tools available in budding yeast are also available in fission yeast in versions taking into account the features of each organism. The delitto perfetto is a powerful approach, initially developed in S. cerevisiae , for in vivo site-directed mutagenesis. Here, we present an adaptation of the approach to S. pombe manipulation and demonstrate its applicability for a rapid, marker-free and efficient in vivo site-directed mutagenesis and N-terminal tagging of nonessential genes in fission yeast.

A new Saccharomyces cerevisiae strain with a mutant Smt3-deconjugating Ulp1 protein is affected in DNA replication and requires Srs2 and homologous recombination for its viability.

The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37 degrees C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.

The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks.

Replication requires homologous recombination (HR) to stabilize and restart terminally arrested forks. HR-mediated fork processing requires single stranded DNA (ssDNA) gaps and not necessarily double strand breaks. We used genetic and molecular assays to investigate fork-resection and restart at dysfunctional, unbroken forks in Schizosaccharomyces pombe. Here, we report that fork-resection is a two-step process regulated by the non-homologous end joining factor Ku. An initial resection mediated by MRN-Ctp1 removes Ku from terminally arrested forks, generating ~110 bp sized gaps obligatory for subsequent Exo1-mediated long-range resection and replication restart. The mere lack of Ku impacts the processing of arrested forks, leading to an extensive resection, a reduced recruitment of RPA and Rad51 and a slower fork-restart process. We propose that terminally arrested forks undergo fork reversal, providing a single DNA end for Ku binding. We uncover a role for Ku in regulating end-resection of unbroken forks and in fine-tuning HR-mediated replication restart.