Nbs1 potentiates ATP-driven DNA unwinding and endonuclease cleavage by the Mre11/Rad50 complex

  1. Tanya T. Paull and
  2. Martin Gellert
  1. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0540 USA

Abstract

The Nijmegen breakage syndrome gene product (Nbs1) was shown recently to associate in vivo with the Mre11 and Rad50 proteins, which play pivotal roles in eukaryotic DNA double-strand break repair, meiotic recombination, and telomere maintenance. We show in this work that the triple complex of recombinant Nbs1, Mre11, and Rad50 proteins binds cooperatively to DNA and forms a distinct protein–DNA species. The Mre11/Rad50/Nbs1 complex displays several enzymatic activities that are not seen without Nbs1, including partial unwinding of a DNA duplex and efficient cleavage of fully paired hairpins. Unwinding and hairpin cleavage are both increased by the presence of ATP. On nonhairpin DNA ends, ATP controls a switch in endonuclease specificity that allows Mre11/Rad50/Nbs1 to cleave a 3′-protruding strand at a double-/single-strand transition. Mutational analysis demonstrates that Rad50 is responsible for ATP binding by the complex, but the ATP-dependent activities are expressed only with Nbs1 present.

Keywords

Footnotes

  • Corresponding author.

  • E-MAIL gellert{at}helix.nih.gov; FAX (301) 496-0201.

    • Received March 15, 1999.
    • Accepted April 12, 1999.
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