Saccharomyces cerevisiae Xrs2 Binds DNA Through Its FHA Domain.

The MRE11-RAD50-NBS1/Xrs2 (MRN/X) complex is a conserved first responder to DNA double-strand breaks (DSBs). All three members of the complex have DNA binding properties that support the range of functions MRN/X performs in its role in DNA DSB repair. Previous structural and functional studies have localized DNA binding sites within MRE11 and RAD50, but no structural model exists for DNA association with NBS1/Xrs2. Here, we identify a DNA binding site within the N-terminal folded FHA-BRCT-BRCT domain of Saccharomyces cerevisiae Xrs2. Using NMR chemical shift perturbations and paramagnetic relaxation enhancements, we define a DNA binding interface on the FHA domain and generate integrative models of the DNA-bound complex via the program HADDOCK. DNA binding overlaps with the site involved in phosphorylated Sae2 peptide binding - an interaction analogous to that between Schizosaccharomyces pombe Nbs1 and phosphorylated Ctp1. Comparative binding assays and site-directed mutagenesis confirm a shared binding surface for DNA and pSae2 on the FHA domain of Xrs2 and highlight the need for functional assays and mutagenesis for validating HADDOCK models. Finally, NMR relaxation experiments reveal altered ps-ns timescale dynamics but unaltered µs-ms conformational exchange upon ligand binding. These findings define a direct DNA binding role for Xrs2 and provide a structural framework for understanding its dual recognition of DNA and phosphoprotein partners during DSB repair.