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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.
Highlights
Homologous recombination plays key roles in double-strand break repair, rescue, and repair of stalled replication forks and meiosis
Our work reveals features of base triplet stepping that are conserved among these three phylogenetic lineages of the Rad51/RecA family and reveals lineage-specific behaviors reflecting properties that are unique to each recombinase
Unbound proteins are flushed away, and double-stranded DNA (dsDNA)-binding activity of the pre-synaptic complexes is probed by the addition of short (70-bp) dsDNA oligonucleotides bearing a single tract of sequence microhomology 8 –15 nucleotides in length that is complementary to a unique location on the M13 single-stranded DNA (ssDNA) [28, 29]
Summary
We used ssDNA curtains and total internal reflection fluorescence microscopy to visualize RecA, Rad, or Dmc presynaptic complexes. Unbound proteins are flushed away, and dsDNA-binding activity of the pre-synaptic complexes is probed by the addition of short (70-bp) dsDNA oligonucleotides bearing a single tract of sequence microhomology 8 –15 nucleotides (nt) in length that is complementary to a unique location on the M13 ssDNA [28, 29]. We sought to determine whether similar results would be obtained if we instead extended the tract of microhomology in the 3Ј 3 5Ј direction (Fig. 2A) To address this question, we used a series of 70-bp oligonucleotides labeled at one end with a single Atto565 dye and bearing
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