4 Zakharyevich, K. et al. Temporally and biochemically distinct activities of Exo1 during meiosis: double-strand break resection and resolution of double Holliday junctions.

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IPdeficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.

No abstract

32During prophase of the first meiotic division, cells deliberately break their DNA. 33These DNA breaks are repaired by homologous recombination, which facilitates 34proper chromosome segregation and enables reciprocal exchange of DNA seg-35 ments between homologous chromosomes, thus promoting genetic diversity in 36 the progeny 1 . A successful completion of meiotic recombination requires nucleo-37 lytic processing of recombination intermediates. Genetic and cellular data impli-38 cated a pathway dependent on the putative MLH1-MLH3 (MutLγ) nuclease in gen-39 erating crossovers, but mechanisms that lead to its activation were unclear 2-4 . 40Here, we have biochemically reconstituted key elements of this pro-crossover 41 pathway. First, we show that human MSH4-MSH5 (MutSγ), which was known to 42 support crossing over [5][6][7] , binds branched recombination intermediates and phys-43 ically associates with MutLγ. This helps stabilize the ensemble at joint molecule 44 structures and adjacent dsDNA. Second, we show that MutSγ directly stimulates 45 DNA cleavage by the MutLγ endonuclease, which demonstrates a novel and unex-46 pected function for MutSγ in triggering crossing-over. Third, we find that MutLγ 47 tion of yeast MutLγ is dependent on the integrity of the metal binding 75 DQHA(X)2E(X)4E motif within Mlh3, implicating the nuclease of Mlh3 in resolving 76 recombination intermediates 2,3,18,19,56 . Despite wealth of genetic and cellular data, 77 the mechanisms that control the MutLγ nuclease and lead to biased joint molecule 78 processing remained undefined. 79 80 81 82 83 84 4 Results 85 86 Human MutLγ is an ATP-stimulated endonuclease 87To study human MutLγ (hMLH1-hMLH3), we expressed and purified the hetero-88 dimer from insect cells ( Fig. 1a and Extended Data Fig. 1a,b). Similarly to the mis-89 match repair (MMR)-specific hMutLα (hMLH1-hPMS2) 20 , the hMLH1-hMLH3 90 complex non-specifically nicked double-stranded supercoiled DNA (scDNA) in the 91 presence of manganese without any other protein co-factor ( Fig. 1b,c, Extended 92 Data Fig. 1c), while almost no activity was observed with magnesium (Extended 93 Data Fig. 1d), which is believed to be the specific metal co-factor 20 . Mutations in 94 the conserved metal binding motif of hMLH3 abolished the endonuclease, indicat-95 ing that the DNA cleavage activity was intrinsic to the hMutLγ heterodimer ( Fig. 96 1d, see also Extended Data Fig. 1e). ATP promoted the nuclease activity >2-fold 97 ( Fig. 1d,e, Extended Data Fig. 1f-h). Experiments with various ATP analogs re-98 vealed that ATP hydrolysis by hMLH1-hMLH3 was required for the maximal stim-99 ulation of DNA cleavage (Fig. 1f, Extended Data Fig. 1h). The N-termini of both 100 hMLH1 and hMLH3 proteins contain conserved Walker motifs implicated in ATP 101 binding and hydrolysis 21 . To define whether the ATPase of hMLH1, hMLH3 or 102 both subunits of the heterodimer promotes its nucleolytic activity, we prepared 103 the respective hMutLγ variants with mutations in the conserved motifs of either 104 subu...

The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA variants are compromised to stimulate Dna2 in the processing of DNA breaks. The results explain phenotypes of replication-proficient but radiation-sensitive RPA mutants and illustrate the unprecedented functional interplay of RPA and Dna2.

SMARCAL1, ZRANB3 and HLTF are all required for the remodeling of replication forks upon stress. Using reconstituted reactions, we show that the motor proteins have unequal biochemical capacities, explaining why they have non-redundant functions. Whereas SMARCAL1 uniquely anneals RPA-coated ssDNA, suggesting an initial function in fork reversal, it becomes comparatively inefficient in subsequent branch migration. We also show that low concentrations of RAD51 and the RAD51 paralog complex, RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2), directly stimulate SMARCAL1 and ZRANB3 but not HLTF, providing a mechanism underlying previous cellular data implicating these factors in fork reversal. Upon reversal, RAD51 protects replication forks from degradation by MRE11, DNA2 and EXO1 nucleases. We show that the protective function of RAD51 unexpectedly depends on its binding to double-stranded DNA, and higher RAD51 concentrations are required for DNA protection compared to reversal. Together, we define the non-canonical functions of RAD51 and its paralogs in replication fork reversal and protection.

No abstract

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition.Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IP's ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork progression in response to treatment with crosslinking agents. Collectively, our work identifies MCM8IP as a key regulator of DNA damage-associated DNA synthesis during DNA recombination and replication.