Rad50 ATPase Activity is Regulated by DNA Ends and Requires Coordination of Both Active Sites

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2017 Apr 4

PMID 28369545

Citations 18

Authors

Rajashree A Deshpande

Ji-Hoon Lee

Tanya T Paull

Affiliations

Soon will be listed here.

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.

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References

Paull T, Gellert M . Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex. Genes Dev. 1999; 13(10):1276-88. PMC: 316715. DOI: 10.1101/gad.13.10.1276. View

Paques F, Haber J . Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 1999; 63(2):349-404. PMC: 98970. DOI: 10.1128/MMBR.63.2.349-404.1999. View

Luo G, Yao M, Bender C, Mills M, Bladl A, Bradley A . Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. Proc Natl Acad Sci U S A. 1999; 96(13):7376-81. PMC: 22093. DOI: 10.1073/pnas.96.13.7376. View

Stewart G, Maser R, Stankovic T, Bressan D, Kaplan M, Jaspers N . The DNA double-strand break repair gene hMRE11 is mutated in individuals with an ataxia-telangiectasia-like disorder. Cell. 1999; 99(6):577-87. DOI: 10.1016/s0092-8674(00)81547-0. View

Hopfner K, Karcher A, Shin D, Craig L, Arthur L, Carney J . Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily. Cell. 2000; 101(7):789-800. DOI: 10.1016/s0092-8674(00)80890-9. View

Zhu J, Petersen S, Tessarollo L, Nussenzweig A . Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. Curr Biol. 2001; 11(2):105-9. DOI: 10.1016/s0960-9822(01)00019-7. View

Rattray A, McGill C, Shafer B, Strathern J . Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1. Genetics. 2001; 158(1):109-22. PMC: 1461648. DOI: 10.1093/genetics/158.1.109. View

Hopfner K, Karcher A, Craig L, Woo T, Carney J, Tainer J . Structural biochemistry and interaction architecture of the DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Cell. 2001; 105(4):473-85. DOI: 10.1016/s0092-8674(01)00335-x. View

Trujillo K, Sung P . DNA structure-specific nuclease activities in the Saccharomyces cerevisiae Rad50*Mre11 complex. J Biol Chem. 2001; 276(38):35458-64. DOI: 10.1074/jbc.M105482200. View

10.

Fukushima T, Takata M, Morrison C, Araki R, Fujimori A, Abe M . Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair. J Biol Chem. 2001; 276(48):44413-8. DOI: 10.1074/jbc.M106295200. View

11.

Lobachev K, Gordenin D, Resnick M . The Mre11 complex is required for repair of hairpin-capped double-strand breaks and prevention of chromosome rearrangements. Cell. 2002; 108(2):183-93. DOI: 10.1016/s0092-8674(02)00614-1. View

12.

Allen C, Kurimasa A, Brenneman M, Chen D, Nickoloff J . DNA-dependent protein kinase suppresses double-strand break-induced and spontaneous homologous recombination. Proc Natl Acad Sci U S A. 2002; 99(6):3758-63. PMC: 122597. DOI: 10.1073/pnas.052545899. View

13.

Hopfner K, Craig L, Moncalian G, Zinkel R, Usui T, Owen B . The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair. Nature. 2002; 418(6897):562-6. DOI: 10.1038/nature00922. View

14.

Chan D, Chen B, Prithivirajsingh S, Kurimasa A, Story M, Qin J . Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev. 2002; 16(18):2333-8. PMC: 187438. DOI: 10.1101/gad.1015202. View

15.

de Jager M, Wyman C, van Gent D, Kanaar R . DNA end-binding specificity of human Rad50/Mre11 is influenced by ATP. Nucleic Acids Res. 2002; 30(20):4425-31. PMC: 137138. DOI: 10.1093/nar/gkf574. View

16.

Hopfner K, Tainer J . Rad50/SMC proteins and ABC transporters: unifying concepts from high-resolution structures. Curr Opin Struct Biol. 2003; 13(2):249-55. DOI: 10.1016/s0959-440x(03)00037-x. View

17.

Ding Q, Reddy Y, Wang W, Woods T, Douglas P, Ramsden D . Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair. Mol Cell Biol. 2003; 23(16):5836-48. PMC: 166339. DOI: 10.1128/MCB.23.16.5836-5848.2003. View

18.

Lee J, Ghirlando R, Bhaskara V, Hoffmeyer M, Gu J, Paull T . Regulation of Mre11/Rad50 by Nbs1: effects on nucleotide-dependent DNA binding and association with ataxia-telangiectasia-like disorder mutant complexes. J Biol Chem. 2003; 278(46):45171-81. DOI: 10.1074/jbc.M308705200. View

19.

Trujillo K, Roh D, Chen L, Van Komen S, Tomkinson A, Sung P . Yeast xrs2 binds DNA and helps target rad50 and mre11 to DNA ends. J Biol Chem. 2003; 278(49):48957-64. DOI: 10.1074/jbc.M309877200. View

20.

Uziel T, Lerenthal Y, Moyal L, Andegeko Y, Mittelman L, Shiloh Y . Requirement of the MRN complex for ATM activation by DNA damage. EMBO J. 2003; 22(20):5612-21. PMC: 213795. DOI: 10.1093/emboj/cdg541. View