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

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.

20 Years of Mre11 Biology: No End in Sight

“…Biochemical analysis of Mre11 nuclease activity confirms that ATP hydrolysis by Rad50 is a prerequisite for Mre11/Rad50mediated nuclease activity on double-stranded DNA (Connelly et al, 1997;Deshpande et al, 2017;Herdendorf et al, 2011;Hopfner et al, 2000a;Paull and Gellert, 1999;Trujillo and Sung, 2001). However, single-stranded DNA with secondary structure, such as M13 phage DNA, can be cleaved by Mre11/ Rad50 complexes in the absence of ATP (Connelly and Leach, 1996;Herdendorf et al, 2011;Hopfner et al, 2000a), and eukaryotic Mre11 exhibits 3 0 to 5 0 exonuclease activity in the absence of Rad50 (Paull and Gellert, 1998;Trujillo et al, 1998), indicating that Rad50-catalyzed ATP hydrolysis is not essential for all Mre11 functions.…”

“…Nbs1 binds to Mre11 through an Mre11-interacting region in the C terminus, which is crystallized with S. pombe Mre11 on the surface of the phosphodiesterase domain and at the Mre11 dimer interface (Schiller et al, 2012). Human Nbs1 is required to promote Mre11 endonuclease activity on blocked DNA ends and hairpin substrates and is important for the stimulatory effect of DNA ends on the rate of Rad50 ATP hydrolysis (Deshpande et al, 2016(Deshpande et al, , 2017Paull and Gellert, 1999). Nbs1 also restrains MR-catalyzed 3 0 to 5 0 exonuclease activity on open DNA ends while promoting 3 0 to 5 0 exonuclease activity at protein-blocked ends (Deshpande et al, 2016); thus, Nbs1 is a key determinant of the outcomes of Mre11 activity.…”

DNA-dependent protein kinase promotes DNA end processing by MRN and CtIP

“…The extreme sensitivity of U2OS cancer cells to simultaneous use of DNA-PKcs and Mre11 inhibitors points toward a potential use of these small molecule inhibitors in treatment of cancers with active homologous recombination pathways. were expressed and purified from Sf21 insect cells as described previously (8,21,24). 3XFLAG-MRN, 3XHA-Ku70/80 heterodimer, and Exo1-biotin were made as described previously (13).…”

The bacterial Mre11–Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions

“…Human and yeast MRN/X have ATPase rates <0.1 ATP/min, which is stimulated 20-fold for human MRN and 10-fold for yeast MRX by linear double-stranded DNA (dsDNA) ( 47 , 48 ). In the absence of DNA, we obtained a similarly low ATPase activity with a k cat of 0.008 s −1 for SbcCD.…”