Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites

Nucleic Acids Res. 2017 May 19;45(9):5255-5268. doi: 10.1093/nar/gkx173.

Abstract

The Mre11-Rad50-Nbs1(Xrs2) (MRN/X) complex is critical for the repair and signaling of DNA double strand breaks. The catalytic core of MRN/X comprised of the Mre11 nuclease and Rad50 adenosine triphosphatase (ATPase) active sites dimerizes through association between the Rad50 ATPase catalytic domains and undergoes extensive conformational changes upon ATP binding. This ATP-bound 'closed' state promotes binding to DNA, tethering DNA ends and ATM activation, but prevents nucleolytic processing of DNA ends, while ATP hydrolysis is essential for Mre11 endonuclease activity at blocked DNA ends. Here we investigate the regulation of ATP hydrolysis as well as the interdependence of the two functional active sites. We find that double-stranded DNA stimulates ATP hydrolysis by hMRN over ∼20-fold in an end-dependent manner. Using catalytic site mutants to create Rad50 dimers with only one functional ATPase site, we find that both ATPase sites are required for the stimulation by DNA. MRN-mediated endonucleolytic cleavage of DNA at sites of protein adducts requires ATP hydrolysis at both sites, as does the stimulation of ATM kinase activity. These observations suggest that symmetrical engagement of the Rad50 catalytic head domains with ATP bound at both sites is important for MRN functions in eukaryotic cells.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Catalytic Domain*
  • DNA / metabolism*
  • DNA Repair Enzymes / chemistry*
  • DNA Repair Enzymes / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Hydrolysis
  • Multiprotein Complexes / metabolism
  • Protein Binding
  • Protein Multimerization

Substances

  • DNA-Binding Proteins
  • Multiprotein Complexes
  • Adenosine Triphosphate
  • DNA
  • Ataxia Telangiectasia Mutated Proteins
  • Rad50 protein, human
  • DNA Repair Enzymes