Open AccessVisualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET
Author(s) -
Brian Can,
Jeffrey Kuhnlein,
Soo-Hyun Yang,
Anita Cheng,
Detlev Schindler,
Jeremy M. Stark,
Rick Russell,
Tanya T. Paull
Publication year - 2013
Publication title -
proceedings of the national academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.1309816110
Subject(s) - rad50 , dna , dna repair , förster resonance energy transfer , microbiology and biotechnology , biophysics , biology , chemistry , genetics , dna binding protein , physics , gene , transcription factor , fluorescence , quantum mechanics
The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.
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