Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase.

DNA damage tolerance (DDT), activated by replication stress during genome replication, is mediated by translesion synthesis and homologous recombination (HR). Here we uncover that DDK kinase, essential for replication initiation, is critical for replication-associated recombination-mediated DDT. DDK relies on its multi-monoSUMOylation to facilitate HR-mediated DDT and optimal retention of Rad51 recombinase at replication damage sites. Impairment of DDK kinase activity, reduced monoSUMOylation and mutations in the putative SUMO Interacting Motifs (SIMs) of Rad51 impair replication-associated recombination and cause fork uncoupling with accumulation of large single-stranded DNA regions at fork branching points. Notably, genetic activation of salvage recombination rescues the uncoupled fork phenotype but not the recombination-dependent gap-filling defect of DDK mutants, revealing that the salvage recombination pathway operates preferentially proximal to fork junctions at stalled replication forks. Overall, we uncover that monoSUMOylated DDK acts with Rad51 in an axis that prevents replication fork uncoupling and mediates recombination-dependent gap-filling.

Parental histone deposition on the replicated strands promotes error-free DNA damage tolerance and regulates drug resistance.

Ctf4 is a conserved replisome component with multiple roles in DNA metabolism. To investigate connections between Ctf4-mediated processes involved in drug resistance, we conducted a suppressor screen of ctf4Δ sensitivity to the methylating agent MMS. We uncovered that mutations in Dpb3 and Dpb4 components of polymerase ε result in the development of drug resistance in ctf4Δ via their histone-binding function. Alleviated sensitivity to MMS of the double mutants was not associated with rescue of ctf4Δ defects in sister chromatid cohesion, replication fork architecture, or template switching, which ensures error-free replication in the presence of genotoxic stress. Strikingly, the improved viability depended on translesion synthesis (TLS) polymerase-mediated mutagenesis, which was drastically increased in ctf4 dpb3 double mutants. Importantly, mutations in Mcm2-Ctf4-Polα and Dpb3-Dpb4 axes of parental (H3-H4)2 deposition on lagging and leading strands invariably resulted in reduced error-free DNA damage tolerance through gap filling by template switch recombination. Overall, we uncovered a chromatin-based drug resistance mechanism in which defects in parental histone transfer after replication fork passage impair error-free recombination bypass and lead to up-regulation of TLS-mediated mutagenesis and drug resistance.

Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites.

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.

The Mgs1/WRNIP1 ATPase is required to prevent a recombination salvage pathway at damaged replication forks.

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.