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SMC Complexes pp 269-287 | Cite as

Purification and Biophysical Characterization of the Mre11-Rad50-Nbs1 Complex

  • Logan R. Myler
  • Michael M. Soniat
  • Xiaoming Zhang
  • Rajashree A. Deshpande
  • Tanya T. Paull
  • Ilya J. FinkelsteinEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

Abstract

The Mre11-Rad50-Nbs1 (MRN) complex coordinates the repair of DNA double-strand breaks, replication fork restart, meiosis, class-switch recombination, and telomere maintenance. As such, MRN is an essential molecular machine that has homologs in all organisms of life, from bacteriophage to humans. In human cells, MRN is a >500 kDa multifunctional complex that encodes DNA binding, ATPase, and both endonuclease and exonuclease activities. MRN also forms larger assemblies and interacts with multiple DNA repair and replication factors. The enzymatic properties of MRN have been the subject of intense research for over 20 years, and more recently, single-molecule biophysics studies are beginning to probe its many biochemical activities. Here, we describe the methods used to overexpress, fluorescently label, and visualize MRN and its activities on single molecules of DNA.

Key words

DNA curtains Single-molecule imaging Homologous recombination DNA repair MRN 

Notes

Acknowledgments

We are indebted to Dr. Mauro Modesti for reagents. This work was supported by CPRIT (to I.J.F.), the National Institutes of Health (GM120554 and CA092584 to I.J.F.) and the Welch Foundation (F-l808 to I.J.F.). M.M.S. is supported by a postdoctoral fellowship, PF-17-169-01-DMC, from the American Cancer Society. L.R.M. is supported by the National Cancer Institute (CA212452). T.T.P. is an investigator of the Howard Hughes Medical Institute. I.J.F. is a CPRIT Scholar in cancer research.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Logan R. Myler
    • 1
  • Michael M. Soniat
    • 1
    • 2
  • Xiaoming Zhang
    • 1
    • 3
  • Rajashree A. Deshpande
    • 1
    • 3
  • Tanya T. Paull
    • 1
    • 3
  • Ilya J. Finkelstein
    • 1
    • 2
    Email author
  1. 1.Department of Molecular Biosciences and Institute for Cellular and Molecular BiologyThe University of Texas at AustinAustinUSA
  2. 2.Center for Systems and Synthetic BiologyThe University of Texas at AustinAustinUSA
  3. 3.The Howard Hughes Medical InstituteThe University of Texas at AustinAustinUSA

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