The Studies On The Function And Mechanism Of Chromatin Remodeller BRG1 In DNA Double-strand Breaks Repair

Genome carries all the genetic information. The stability of genome is critical for the organism survival and reproduction. However, the environment around the DNA is not absolute safe. There are many factors that can induce DNA damage, such as cell metabolism by-products, replication fork collapse, extra-cellular toxic stimulis and so on. DNA damage if unrepaired or misrepaired, can lead to genetic mutation which may do harm to organism. In order to maintain the genome stability, the eukaryotic DNA is packaged into complicated and condensed chromatin structure by histones. The structure is a two-side sword. On one hand, it can prevent genome from various toxic factors; on the other hand, it is a big barrier for DNA metabolism, such as DNA transcription, replication and repair. Hence, the dynamic of chromatin plays an important role in DNA metabolism.DNA double-strand breaks(DSBs) is a type of lethal DNA damage. To concer the DNA damage, organism has evolved a complicated and well-organized DNA repair mechanism. DNA repair requires tight coordination among the factors modulating chromatin structure, cell cycle regulator, cell skeleton modulator, and the DNA repair machinery. BRG1 is the core subunit of the chromatin remodelling complex SWI/SNF. It is an ATPase enzyme. Previous studies have showed that BRG1 is often linked to tumorigenesis and genome instability, and its role in DSBs repair remains largely unclear.In the present study, we first built ETO-, bleomycin-, and laser-induced DSBs repair model in vitro. Cell colony survival assay showed that BRG1 depletion increases cell sensitivity to DNA damage agents and decreases the viability of damaged cell. Furthermore, cell comet assay and immunofluorescence demonstrated that BRG1 is critical for DSBs repair. We also found that BRG1 accumulates at DNA damage sites by Ch IP assay on AsiSI-induced DSBs and chromatin fraction assay. There are two major processes for the DNA DSBs repair in mammalian cells: homologous recombination(HR) and nonhomologous end-joining(NHEJ). Using DR-GFP and EJ5-GFP reporter systems, we demonstrated that BRG1 facilitates homologous recombination repair rather than nonhomologous end-joining(NHEJ) repair. What’s more, immunofluorescence, co-immunoprecipitation assay and time-lapse live cell microscope all indicated that BRG1 regulates the recruitment of RAD51 onto single-stranded DNAs(ssDNAs) to initiate homologous DNA strand invasion and this function is mediated by the interaction between BRG1 and RAD52. Our present study provides a mechanistic insight into how BRG1 plays a substantial role in the homologous recombination repair pathway in mammalian cells and underscores the tight link between chromatin structure and DNA metabolism.