Structure of the ATP-Free Mre11-Rad50 DNA Damage Repair Complex Bound to DNA Substrates

Abstract

DNA double strand breaks, where both strands of the double-stranded DNA are broken, are among the most harmful forms of DNA damage because an intact template is not present for repair. The Mre11-Rad50–Nbs1 (Xrs2 in yeast) complex plays a central and critical role in the detection and repair of DSBs and is conserved in all kingdoms of life, as Mre11- Rad50 (MR) in prokaryotes and as MRN/X in eukaryotes. Although there are some structural models available for MR complex bound to DNA substrates, there are limitations in these structures. Therefore, our goal is to examine the MR complex bound to different DNA substrates using an integrated structural biology approach. Using uniformly deuterated, 13CH3-methyl labeled samples, we measured distance restraints for the ∼120 kDa P. furiosus MR complex bound to spin-labeled double-stranded or single-stranded DNA substrates via paramagnetic relaxation enhancements (PREs). Additional distance and global restraints were measured from Luminescence Resonance Energy Transfer (LRET) and Small Angle X-ray Scatter (SAXS) experiments. These data then served as restraints for calculating structural models using multi-body docking implemented in HADDOCK. Our models revealed novel modes of DNA binding for the ATP-free MR complex and provide the first depiction of a single-stranded DNA-bound complex. We tested our models through biochemical activity assays to determine the effect of disruption of key interactions, via mutation, in our models.