Slow extension of the invading DNA strand in a D-loop formed by RecA-mediated homologous recombination may enhance recognition of DNA homology

Daniel Lu,Claudia Danilowicz,Tommy F Tashjian,Chantal Prévost,Veronica G Godoy,Mara Prentiss

doi:10.1074/jbc.ra119.007554

Abstract

DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks. However, the process of recombinational repair between short DNA regions of accidental similarity can lead to fatal genomic rearrangements. Previous studies have probed how effectively RecA discriminates against interactions involving a short similar sequence that is embedded in otherwise dissimilar sequences but have not yielded fully conclusive results. Here, we present results of in vitro experiments with fluorescent probes strategically located on the interacting DNA fragments used for recombination. Our findings suggest that DNA synthesis increases the stability of the recombination products. Fluorescence measurements can also probe the homology dependence of the extension of invading DNA strands in D-loops formed by RecA-mediated strand exchange. We examined the slow extension of the invading strand in a D-loop by DNA polymerase (Pol) IV and the more rapid extension by DNA polymerase LF-Bsu. We found that when DNA Pol IV extends the invading strand in a D-loop formed by RecA-mediated strand exchange, the extension afforded by 82 bp of homology is significantly longer than the extension on 50 bp of homology. In contrast, the extension of the invading strand in D-loops by DNA LF-Bsu Pol is similar for intermediates with ≥50 bp of homology. These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase.

Highlights

DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks
These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase
If heteroduplex stability is influenced by the extension of the invading strand in a D-loop, in vitro studies that do not include DNA polymerase may not capture the N probability of permanently incorporating regions of accidental homology into genomes

Summary

Edited by Patrick Sung

DNA recombination resulting from RecA-mediated strand exchange aided by RecBCD proteins often enables accurate repair of DNA double-strand breaks. The extension of the invading strand in D-loops by DNA LF-Bsu Pol is similar for intermediates with >50 bp of homology These results suggest that fatal genomic rearrangements due to the recombination of small regions of accidental homology may be reduced if RecA-mediated strand exchange is immediately followed by DNA synthesis by a slow polymerase. That previous work did not consider how DNA synthesis that extends the invading strand in a D-loop affects the yield of heteroduplex products or how N influences the progress of invading strand extension after the extension is triggered by the formation of a heteroduplex product [19]. DNA Pol IV is up-regulated by the SOS response that follows formation of a double-strand break among other insults [20], and DNA Pol IV is known to extend invading strands in D-loops formed by RecA-mediated homologous recombination [9]. We present a model that could explain these differences in homology dependence and consider implications of the model for genomic rearrangements

Results

Discussion

Strand exchange and DNA synthesis assay

Preparation of dsDNA samples

FRET measurements