Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with ␥-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.Genome stability relies on the accurate repair of doublestrand breaks (DSBs) that arise naturally during DNA replication or from treatment with exogenous DNA-damaging agents. DSBs in human cells may be repaired by nonhomologous end joining or homologous recombination. The MRE11-RAD50-NBS1 (MRN) complex has essential functions in numerous facets of genome stability. During the cell cycle, MRN is associated to chromatin, which is consistent with a role in the surveillance of genome integrity. Chromatin association is not increased following ionizing radiation (41). However, a redistribution of the MRN complex from the chromatin to sites of DNA damage occurs. When cells are challenged with ionizing radiation, ataxia-telangiectasia mutated protein (ATM) phosphorylates histone H2AX (30), a key event in this process. The phosphorylation of H2AX is thought to recruit MRN directly to DSBs, since NBS1 interacts directly with phosphorylated H2AX (17). MDC1 is also a key molecular linker responsible for bridging NBS1 with phosphoepitopes generated in DSBflanking chromatin, as MRN components do not form foci in the absence of MDC1 (22). MRN is a central player during checkpoint signaling of DNA damage. MRN stimulates ATM kinase activity on p53, CHK2, and H2AX (19). ATM phosphorylates NBS1 on serine 343, which is necessary for the proper S-phase checkpoint following ionizing radiation (21). Consistent with key functions in DNA damage signaling and repair, ataxia-telangiectasia-like disorder (34) and Nijmegen breakage syndrome (40), which are caused by mutations in MRE11 and NBS1, respectively, are characterized by an increased sensitivity to ionizing radiation, checkpoint problems, and the accumulation of DSBs and aberrant chromosomes, leading to cancer.DNA repair functions of the MRN complex involve the processing of DNA ends during homologous recombination, nonhomologous end joining, and maintenance of telomere...