Molecular Mechanism For The SRCAP Chromatin-remodeling Factor In Promoting Homologous Recombination Repair

It is estimated that each cell encounters up to105spontaneous DNA lesions arising from endogenous and exogenous genotoxic stresses per day. DNA double-strand breaks (DSBs) are the most toxic DNA lesions among them. Unrepaired or incorrectively repaired DNA may cause cell death, mutations, genomic instability or cancer. In response, two major pathways including homologous recombination (HR) and non-homologous end joining (NHEJ) have evolved to cope with DSBs. HR is a high-fidelity repair pathway that utilizes the homologous sequence as template to repair the broken DNA. Conversely, NHEJ just ligates truncated DNA ends together with no requirement for intrastrand homology, thus it is believed to be an error-prone pathway. HR thereby is a critical pathway to maintain genomic stability. DNA-end resection is the initial step of homologous recombination repair. In vertebrates, MRE11-RAD50-NBS1(MRN) complex cooperates with CtIP protein.to initiate the5’â†'3’ssDNA limited resection at the break ends. While genomic DNA is in the densely-folded structure of chromatin with nucleosome as its basic unit, such organizational structure of DNA is a natural barrier of DSB end resection, so that the DNA structural relax mediated by chromatin remodeling factors becomes an important step to initiate DNA-end resection effectively, but we know little about chromatin remodeling factors incurred by DSB end resection initiation process. SRCAP is an ATP-dependent chromatin-remodeling factor in INO80family, and SRCAP mutation is an important cause resulting in Floating-Harbor syndrome, but it is not clear about the functions of SRCAP during DNA repair. Therefore, we hypothesis that SRCAP involve in the process of DSBs repairs by modulating chromatin.Firstly, we verified whether SRCAP participated in DSBs repair directly, Our experimental results showed that the SRCAP deletion significantly decreased cell survival rate and DNA repairing efficiency upon exposure to DNA damage-inducing agents. Further study showed that SRCAP could be recruited to DSB site to participate in the repair process directly. HR and NHEJ are two major pathways for DSBs repair. In HR report analysis system, we found that SRCAP depletion dramatically decreased HR frequency. In line with these results, the recruitment of RAD51, an indispensable HR recombinase, was found defective at DNA damage sites in SRCAP-depleted cells. Therefore, these data indicated that SRCAP promotes DSBs repair through HR pathway. DNA-end resection is a key step for the initiation of homologous recombination repair. Thus, we examined whether the SRCAP depletion affects DNA-end resection. As expected, SRCAP depletion severely impaired the DSB end resection to generate3’ssDNA, and3’ssDNA also plays a critical role in the activation of the ATR/CHK1pathway to promote cell cycle arrest. Indeed, the loss of SRCAP impaired the phosphorylate activation of CHK1and RPA2induced by CPT and IR. These data demonstrate that SRCAP promoted DNA-end resection further facilitated the initiation of HR repair. Meanwhile, the ATR-dependent checkpoint activation induced arrest cell cycle provides necessary time for the HR.The DNA-end resection is initiated by MRN complex and the associated CtIP protein. We next assessed whether SRCAP depletion affects the DNA damage dependent recruitment of these factors. Interestingly, SRCAP depletion dramatically impaired the accumulation of CtIP to damaged DNA, but not NBS1. These data therefore indicated that SRCAP facilitates DNA-end resection by promoting CtIP accumulation at DSB sites. Successively, we further tried to decipher how SRCAP promotes the recruitment of CtIP to the DSB damage site. The co-immunoprecipitation assay showed that SRCAP physically interacts with CtIP. Furthermore, our results indicate that residues36-50of CtIP are required not only for SRCAP binding but also for the efficient recruitment of CtIP at DSBs. Therefore, SRCAP recruits CtIP to initiate resection of the DSB ends by the physical SRCAP-CtIP interaction.Moreover, SRCAP is an ATP-dependent chromatin remodeling factor, In light of this, we next examined whether SRCAP altered chromatin structure will promote the efficient recruitment of CtIP at DSBs. Our results revealed that SRCAP depletion made cell chromatin structure even more compactly folded status. Significantly, chromatin relaxation reagent could restore the chromatin structure back into normal in the SRCAP knocking down cells, and also partially rescue the SRCAP depletion induced defects in the recruitment of CtIP at DSBs. Furthermore, the SRCAP-depletion induced defects in chromatin relaxation and CtIP recruitment could not be rescued by ectopic expression of an ATPase-dead mutant of SRCAP. These results suggest that SRCAP may facilitate CtIP recruitment through promoting chromatin relaxation in an ATP-dependent manner.Taken together, SRCAP can be rapidly recruited to damaged chromatin when DSBs occurs, then two mechanism were employed to promote the recruitment of CtIP at DSBs for DNA-end resection:on the one hand, SRCAP relaxes the highly folded chromatin structure in an ATP dependent manner. On the other hand, it recruits CtIP by a direct physical interaction, so as to promote ATR/CHK1checkpoint activation and homologous recombination repair.