BRG1 promotes DNA double-strand break repair by facilitating the replacement of RPA with RAD51

Abstract

DNA double-strand breaks (DSBs) are a type of lethal DNA damage. The repair of DSBs requires tight coordination between the factors modulating chromatin structure and the DNA repair machinery. BRG1, the ATPase subunit of the chromatin remodelling complex Switch/Sucrose non-fermentable (SWI/SNF), is often linked to tumorigenesis and genome instability, and its role in DSB repair remains largely unclear. In the present study, we show that BRG1 is recruited to DSB sites and enhances DSB repair. Using DR-GFP and EJ5-GFP reporter systems, we demonstrate that BRG1 facilitates homologous recombination repair rather than nonhomologous end-joining (NHEJ) repair. Moreover, the BRG1-RAD52 complex mediates the replacement of RPA with RAD51 on single-stranded DNA (ssDNA) to initiate DNA strand invasion. Loss of BRG1 results in a failure of RAD51 loading onto ssDNA, abnormal homologous recombination repair and enhanced DSBinduced lethality. Our present study provides a mechanistic insight into how BRG1, which is known to be involved in chromatin remodelling, plays a substantial role in the homologous recombination repair pathway in mammalian cells.

RB localizes to DNA double-strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1

“…BRG1 is a catalytic subunit of the SWI/SNF family of ATPases that has been shown to interact with RB in the context of transcriptional repression (Dunaief et al 1994;Strobeck et al 2000;Kang et al 2004;Liu et al 2004) and also to be recruited to DSBs (Kakarougkas et al 2014;Gong et al 2015;Qi et al 2015). We found that while the BRG1 ATPase is recruited to DSBs in control U2OS cells, it was not recruited in cells depleted for either RB or E2F1 (Fig.…”

“…We show here that the loss of RB results in defective recruitment of the BRG1 ATPase to DSBs, and, accordingly, this defect was also observed in E2f1 S29A/S29A MEFs. While the involvement of BRG1 in DNA damage signaling and repair has been described previously (Kakarougkas et al 2014;Gong et al 2015;Qi et al 2015), the mechanism by which this ATPase is recruited to DSBs has been elusive. It is important to note that the interaction between RB and BRG1 has long been described in the context of transcriptional repression (Dunaief et al 1994;Strobeck et al 2000;Kang et al 2004;Liu et al 2004;Flowers et al 2013).…”

“…Moreover, the HEL1 helicase was shown to promote end resection and HR likely by modifying nucleosomes at the site of the break (Costelloe et al 2012). The BRG1 ATPase has also been shown to be recruited to DSBs to stimulate repair (Gong et al 2015;Qi et al 2015) and also repress transcription in order for efficient repair to take place (Kakarougkas et al 2014). While these and other chromatin remodelers are known to be critical for altering chromatin structure to promote DSB repair, the mechanisms by which these proteins are recruited to sites of DSBs are poorly understood.…”

Regulation of Mec1 kinase activity by the SWI/SNF chromatin remodeling complex

“…Recruitment of ATP-dependent chromatin remodeling complexes to DSBs (Ray et al 2009;Lee et al 2010;Qi et al 2015) and the remodeling of the surrounding chromatin occur on a timescale similar to that of H2A phosphorylation. Moreover, it has been shown that the phosphorylation of Ies4 (a subunit of the INO80 chromatin remodeling complex) by Mec1/ Tel1 in response to DNA damage directs INO80 function toward checkpoint regulation (Morrison et al 2007).…”

β-Actin regulates interleukin 6-induced p21 transcription by interacting with the Rpb5 and Rpb7 subunits of RNA polymerase II

“…Then, 30 μl of protein G beads were added to the lysates and mixed in a shaker at 4°C for 3 h. The beads were washed three times with lysis buffer and eluted by boiling in SDS sample buffer for 5 min. The eluted proteins were detected by immunoblotting with the appropriate antibodies (Qi et al 2015).…”