Nucleic Acids Research Advance Access originally published online on November 25, 2008
Nucleic Acids Research 2009 37(1):158-171; doi:10.1093/nar/gkn914
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Nucleic Acids Research, 2009, Vol. 37, No. 1 158-171
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Loop 2 in Saccharomyces cerevisiae Rad51 protein regulates filament formation and ATPase activity
1Department of Microbiology, University of California, Davis, CA 95616-8665, 2Department of Biochemistry and Molecular Genetics, University of Virginia, Box 800733, Charlottesville, VA 22908 and 3Department of Molecular and Cellular Biology, University of California, Davis, CA 95616-8665, USA
*To whom correspondence should be addressed. Tel: +1 530 752 3001; Fax: +1 530 752 3011; Email: wdheyer{at}ucdavis.edu
Received September 22, 2008. Revised October 16, 2008. Accepted October 29, 2008.
Previous studies showed that the K342E substitution in the Saccharomyces cerevisiae Rad51 protein increases the interaction with Rad54 protein in the two-hybrid system, leads to increased sensitivity to the alkylating agent MMS and hyper-recombination in an oligonucleotide-mediated gene targeting assay. K342 localizes in loop 2, a region of Rad51 whose function is not well understood. Here, we show that Rad51-K342E displays DNA-independent and DNA-dependent ATPase activities, owing to its ability to form filaments in the absence of a DNA lattice. These filaments exhibit a compressed pitch of 81 Å, whereas filaments of wild-type Rad51 and Rad51-K342E on DNA form extended filaments with a 97 Å pitch. Rad51-K342E shows near normal binding to ssDNA, but displays a defect in dsDNA binding, resulting in less stable protein-dsDNA complexes. The mutant protein is capable of catalyzing the DNA strand exchange reaction and is insensitive to inhibition by the early addition of dsDNA. Wild-type Rad51 protein is inhibited under such conditions, because of its ability to bind dsDNA. No significant changes in the interaction between Rad51-K342E and Rad54 could be identified. These findings suggest that loop 2 contributes to the primary DNA-binding site in Rad51, controlling filament formation and ATPase activity.