The Role Of AUNIP In DNA Double-strand Break Repair Pathway Choice

DNA double strand breaks(DSBs)are the most deleterious type of DNA damage,which if unrepaired or repaired incorrectly will lead to mutations,genomic instability and cell death.DSBs can be classfied into two categories based on their nature--two-ended DSBs and replication-associated one-ended DSBs.Two-ended DSBs are usually induced by exogenous DNA damaging agents like X-ray or γ-ray,while chemical agents introducing stalled or collapsed replication forks can generate replication-associated one-ended DSBs.DSBs are mainly repaired by non-homologous end joining(NHEJ)or homologous recombination(HR).NHEJ is a relatively fast process that directly ligates two broken DNA ends.In contrast,HR is a complicated multi-step repair pathway.HR requires CtIP-and MRN(Mre11-RAD50-NBS1)-dependent DNA end resection to generate single-stranded DNA tails that are first coated by the trimeric single-stranded DNA binding protein replication protein A(RPA).Subsequently,the RAD51 recombinase replaces RPA to form RAD51-ssDNA filaments.These filaments then catalyze homology searching,followed by 3’strand invasion,new DNA synthesis,and ligation.Emerging evidence indicated that two-ended DSBs can be repaired by either NHEJ or HR,with the NHEJ is the dominant pathway.In contrast to two-ended DSBs,replication-associated one-ended DSBs are almost exclusively repaired by HR repair pathway because NHEJ of two one-ended DSBs would give rise to translocations and genomic instability.However,how these replication-associated one-ended DSBs are distinguished from these two-ended DSBs in cells is largely unclear.In this study,we used an affinity purification approach to isolate CtIP-associated protein complexes and have identified AUNIP,a largely uncharacterized protein,as the key determinant of DSBs repair pathway choice.On one hand,AUNIP directly interacts with the key resection factor CtIP.On the other hand,AUNIP has intrisic DNA binding ability with a strong preference for DNA substrates that mimic structures generated at stalled replication fork.This DNA binding ability is critical for both AUNIP and its binding partner CtIP recruitment to sites of DNA damage,and thereby for CtIP-dependent DNA end resection and subsequent HR.Accordingly,loss of AUNIP or ablation of its ability to bind to DNA or CtIP compromises CtIP-dependent DNA-end resection and homology-directed repair,and results in cell hypersensitivity towards a variety of DSB-inducing agents,particularly those that induce replication-associated DSBs.We propose that,through enhancing the tethering of CtIP at perturbed replication forks,AUNIP directs replication-associated one-ended DSBs towards the HR repair pathway.