A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response

Abstract

Repair of DNA double strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We performed a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA damage response and pre-mRNA processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions via multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of Rad51 is a common source of RNAi false-positives, sounding a cautionary note for siRNA screens and RNAi-based studies of HR.

Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase

“…Other DSB repair pathways are also detected in primary cell cultures, particularly NHEJ, consistent with the presence of additional pathways to repair this genomethreatening lesion. Although the DR-GFP reporter cannot be used to measure all types of HDR, its use for assaying intrachromosomal gene conversion events and identifying the factors that promote or suppress these events is well-proven (10,40,41); its incorporation into a mouse model now allows for the study of HDR in primary cell types rather than transformed cell lines, which may have altered repair phenotypes (for example, because of altered expression of repair factors like RAD51) (14,15).…”

The RNA-binding Protein Fused in Sarcoma (FUS) Functions Downstream of Poly(ADP-ribose) Polymerase (PARP) in Response to DNA Damage

“…PARP ADP-ribosylation also appears to provide a common mechanism for the recruitment of at least a subset of RNA-processing factors to DNA damage sites including RBMX and NONO (51,52,75). NONO is recruited by virtue of an RRM1 domain-dependent interaction with PAR (52).…”

“…Although other studies have implicated a role for FUS in maintaining resistance to genotoxic stress, possibly via participation in HR repair (34,40,41), the present results indicate that FUS functions are required for optimal DSB repair through both NHEJ and HR. FUS joins a growing list of RNA-processing factors including RBMX, PPM1G, THRAP3, hnRNPUL1 and -2, and NONO, that are recruited to the proximity of DSBs and whose activities are required for genotoxin resistance and DNA repair (51)(52)(53)(54).…”

CTCF prevents genomic instability by promoting homologous recombination-directed DNA double-strand break repair

“…To determine how CTCF functions in the HR, we analyzed X-ray irradiation-induced Rad51 foci formation in the CTCF knockdown cells, since Rad51 is a key repair factor in HR (32)(33)(34), and aggregates into DNA repair foci after ionizing irradiation or DNA damage-inducing drug treatment (35,36). We found that the knockdown decreased the percentage of cells with five or more X-ray-induced Rad51 foci decreased from 42.13% to <10.17% (Fig.…”