The error-free repair of double-strand DNA breaks by homologous recombination (HR) ensures genomic stability using undamaged homologous sequence to copy genetic information. While some of the aspects of the initial steps of HR are understood, the molecular mechanisms underlying events downstream of the D-loop formation remain unclear. Therefore, we have reconstituted D-loop-based in vitro recombination-associated DNA repair synthesis assay and tested the efficacy of polymerases Pol δ and Pol η to extend invaded primer, and the ability of three helicases (Mph1, Srs2 and Sgs1) to displace this extended primer. Both Pol δ and Pol η extended up to 50% of the D-loop substrate, but differed in product length and dependency on proliferating cell nuclear antigen (PCNA). Mph1, but not Srs2 or Sgs1, displaced the extended primer very efficiently, supporting putative role of Mph1 in promoting the synthesis-dependent strand-annealing pathway. The experimental system described here can be employed to increase our understanding of HR events following D-loop formation, as well as the regulatory mechanisms involved.

Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability.

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The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

Strategies to resolve replication blocks are critical for the maintenance of genome stability. Among the factors implicated in the replication stress response is the ATP-dependent endonuclease ZRANB3. Here, we present the structure of the ZRANB3 HNH (His-Asn-His) endonuclease domain and provide a detailed analysis of its activity. We further define PCNA as a key regulator of ZRANB3 function, which recruits ZRANB3 to stalled replication forks and stimulates its endonuclease activity. Finally, we present the co-crystal structures of PCNA with two specific motifs in ZRANB3: the PIP box and the APIM motif. Our data provide important structural insights into the PCNA-APIM interaction, and reveal unexpected similarities between the PIP box and the APIM motif. We propose that PCNA and ATP-dependency serve as a multi-layered regulatory mechanism that modulates ZRANB3 activity at replication forks. Importantly, our findings allow us to interpret the functional significance of cancer associated ZRANB3 mutations.

Stalling of replication forks at unrepaired DNA lesions can result in discontinuities opposite the damage in the newly synthesized DNA strand. Translesion synthesis or facilitating the copy from the newly synthesized strand of the sister duplex by template switching can overcome such discontinuities. During template switch, a new primer–template junction has to be formed and two mechanisms, including replication fork reversal and D-loop formation have been suggested. Genetic evidence indicates a major role for yeast Rad5 in template switch and that both Rad5 and its human orthologue, Helicase-like transcription factor (HLTF), a potential tumour suppressor can facilitate replication fork reversal. This study demonstrates the ability of HLTF and Rad5 to form a D-loop without requiring ATP binding and/or hydrolysis. We also show that this strand-pairing activity is independent of RAD51 in vitro and is not mechanistically related to that of another member of the SWI/SNF family, RAD54. In addition, the 3′-end of the invading strand in the D-loop can serve as a primer and is extended by DNA polymerase. Our data indicate that HLTF is involved in a RAD51-independent D-loop branch of template switch pathway that can promote repair of gaps formed during replication of damaged DNA.