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J Biol Chem. 2017 Jun 30;292(26):11125-11135. doi: 10.1074/jbc.M117.787614. Epub 2017 May 5.

Sequence imperfections and base triplet recognition by the Rad51/RecA family of recombinases.

Author information

1
From the Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 and.
2
the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
3
From the Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032 and ecg2108@cumc.columbia.edu.

Abstract

Homologous recombination plays key roles in double-strand break repair, rescue, and repair of stalled replication forks and meiosis. The broadly conserved Rad51/RecA family of recombinases catalyzes the DNA strand invasion reaction that takes place during homologous recombination. We have established single-stranded (ss)DNA curtain assays for measuring individual base triplet steps during the early stages of strand invasion. Here, we examined how base triplet stepping by RecA, Rad51, and Dmc1 is affected by DNA sequence imperfections, such as single and multiple mismatches, abasic sites, and single nucleotide insertions. Our work reveals features of base triplet stepping that are conserved among these three phylogenetic lineages of the Rad51/RecA family and also reveals lineage-specific behaviors reflecting properties that are unique to each recombinase. These findings suggest that Dmc1 is tolerant of single mismatches, multiple mismatches, and even abasic sites, whereas RecA and Rad51 are not. Interestingly, the presence of single nucleotide insertion abolishes recognition of an adjacent base triplet by all three recombinases. On the basis of these findings, we describe models for how sequence imperfections may affect base triplet recognition by Rad51/RecA family members, and we discuss how these models and our results may relate to the different biological roles of RecA, Rad51, and Dmc1.

KEYWORDS:

DNA repair; Dmc1; Rad51; RecA; homologous recombination; microscopy; protein-DNA interaction; single-molecule biophysics

PMID:
28476890
PMCID:
PMC5491793
DOI:
10.1074/jbc.M117.787614
[Indexed for MEDLINE]
Free PMC Article

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