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Rad50 ATPase activity is regulated by DNA ends and requires coordination of both active sites
Rajashree A Deshpande1, Ji-Hoon Lee1, Tanya T Paull1
1Howard Hughes Medical Institute, Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
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.
Insights
The Mre11-Rad50-Nbs1 (MRN/X) complex requires dual ATP hydrolysis for DNA repair and ATM activation. Both Rad50 ATPase sites are essential for DNA-stimulated hydrolysis and subsequent cellular functions.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Biochemistry
Background:
- The Mre11-Rad50-Nbs1 (MRN/X) complex is crucial for sensing and repairing DNA double-strand breaks.
- The complex's function is regulated by ATP binding and hydrolysis at the Rad50 ATPase sites.
- The interplay between ATP hydrolysis and Mre11 nuclease activity is key to DNA end processing.
Purpose of the Study:
- To investigate the regulation of ATP hydrolysis within the MRN/X complex.
- To determine the interdependence of the two Rad50 ATPase active sites.
- To elucidate the role of dual ATP hydrolysis in MRN/X-mediated DNA repair and signaling.
Main Methods:
- Utilized catalytic site mutants of Rad50 to create dimers with single functional ATPase sites.
- Measured ATP hydrolysis rates of the human MRN (hMRN) complex in the presence and absence of double-stranded DNA.
- Assessed the impact of ATP hydrolysis on DNA endonucleolytic cleavage and ATM kinase activation.
Main Results:
- Double-stranded DNA stimulates hMRN ATP hydrolysis over 20-fold in an end-dependent manner.
- Both Rad50 ATPase sites are required for DNA-stimulated ATP hydrolysis.
- MRN/X-mediated DNA cleavage and ATM activation necessitate ATP hydrolysis at both ATPase sites.
Conclusions:
- Symmetrical engagement of both Rad50 ATPase sites is critical for MRN/X complex function.
- Dual ATP hydrolysis is essential for DNA end processing and ATM signaling in eukaryotic cells.
- This highlights a coordinated mechanism for DNA double-strand break repair.