Strand invasion by HLTF as a mechanism for template switch in fork rescue.

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

RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination

International audienceThe proficiency of cancer cells to repair DNA double-strand breaks (DSBs) by homologous recombination (HR) is a key determinant in predicting response to targeted therapies such as PARP inhibitors. The RAD51 paralogs work as multimeric complexes and act downstream of BRCA1 to facilitate HR. Numerous epidemiological studies have linked RAD51 paralog mutations with hereditary cancer predisposition. Despite their substantial links to cancer, RAD51 paralog HR function has remained elusive. Here we identify isoform 1 as the functional isoform of RAD51D, whereas isoform 4 which has a large N-terminal deletion (including the Walker A motif), and isoform 6 which includes an alternate exon in the N-terminus, are non-functional. To determine the importance of this N-terminal region, we investigated the impact of cancer-associated mutations and SNPs in this variable RAD51D N-terminal region using yeast-2-hybrid and yeast-3-hybrid assays to screen for altered protein-protein interactions. We identified two cancer-associated mutations close to or within the Walker A motif (G96C and G107 V, respectively) that independently disrupt RAD51D interaction with XRCC2. We validated our yeast interaction data in human U2OS cells by co-immunoprecipitation and determined the impact of these mutations on HR-proficiency using a sister chromatid recombination reporter assay in a RAD51D knockout cell line. Our investigation reveals that the interaction of RAD51D with XRCC2 is required for DSB repair. By characterizing the impact of cancer-associated mutations on RAD51D interactions, we aim to develop predictive models for therapeutic sensitivity and resistance in patients who harbor similar mutations in RAD51D