The MRE11 GAR motif regulates DNA double-strand break processing and ATR activation

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• The MRE11/RAD50/NBS1 complex is essential for sensing and responding to DNA double-strand breaks (DSBs).
• Arginine methylation of MRE11 by PRMT1 within its glycine-arginine-rich (GAR) motif is a known post-translational modification.
• The functional significance of MRE11 arginine methylation in DNA repair pathways remains incompletely understood.

Purpose of the Study:

• To investigate the role of arginine methylation in the MRE11 GAR motif in DNA double-strand break repair.
• To elucidate the impact of abrogated MRE11 methylation on cellular responses to DNA damage.
• To establish a mechanistic link between MRE11 post-translational modification and DNA repair signaling.

Main Methods:

• Generation of a mouse knock-in allele (Mre11(RK/RK)) replacing GAR motif arginines with lysines, creating MRE11(RK) protein lacking methylation.
• Phenotypic analysis of Mre11(RK/RK) mice and derived mouse embryonic fibroblasts (MEFs) for sensitivity to gamma irradiation (IR).
• Assessment of cell cycle checkpoint activation, chromosome instability, DNA damage response signaling (ATR/CHK1, ATM), and recruitment of repair proteins (RPA, RAD51) to DSBs.
• In vitro biochemical assays to evaluate the DNA-binding and exonuclease activities of the M(RK)RN complex.

Main Results:

• Mre11(RK/RK) mice exhibited hypersensitivity to gamma irradiation, with associated cell cycle checkpoint defects and chromosome instability.
• MRE11(RK/RK) MEFs showed impaired ATR/CHK1 signaling and defects in RPA and RAD51 recruitment to DNA damage sites.
• The M(RK)RN complex assembled and localized to DSBs, activating the ATM pathway normally.
• In vitro assays revealed exonuclease and DNA-binding defects in the M(RK)RN complex, correlating with impaired DNA end resection and ATR activation in vivo.

Conclusions:

• Genetic evidence confirms the critical role of the MRE11 GAR motif in DNA double-strand break repair.
• Post-translational arginine methylation of MRE11 is mechanistically linked to efficient DNA end resection and DSB processing.
• Disruption of MRE11 methylation impairs ATR/CHK1 checkpoint signaling, underscoring its importance in maintaining genomic integrity.