Nascent DNA Synthesis During Homologous Recombination Is Synergistically Promoted by the Rad51 Recombinase and DNA Homology

Abstract

In this study, we exploited a plasmid-based assay that detects the new DNA synthesis (39 extension) that accompanies Rad51-mediated homology searching and strand invasion steps of homologous recombination to investigate the interplay between Rad51 concentration and homology length. Mouse hybridoma cells that express endogenous levels of Rad51 display an approximate linear increase in the frequency of 39 extension for homology lengths of 500 bp to 2 kb. At values below 500 bp, the frequency of 39 extension declines markedly, suggesting that this might represent the minimal efficient processing segment for 39 extension. Overexpression of wild-type Rad51 stimulated the frequency of 39 extension by 3-fold for homology lengths ,900 bp, but when homology was .2 kb, 39 extension frequency increased by as much as 10-fold. Excess wild-type Rad51 did not increase the average 39 extension tract length. Analysis of cell lines expressing N-terminally FLAG-tagged Rad51 polymerization mutants F86E, A89E, or F86E/A89E established that the 39 extension process requires Rad51 polymerization activity. Mouse hybridoma cells that have reduced Brca2 (Breast cancer susceptibility 2) due to stable expression of small interfering RNA show a significant reduction in 39 extension efficiency; expression of wild-type human BRCA2, but not a BRCA2 variant devoid of BRC repeats 1-8, rescues the 39 extension defect in these cells. Our results suggest that increased Rad51 concentration and homology length interact synergistically to promote 39 extension, presumably as a result of enhanced Brca2-mediated Rad51 polymerization. Homologous recombination is a complex, multi-step process (reviewed in Brugmans et al. 2007). In eukaryotes, the central step in the repair of a DNA DSB involves the binding of multiple monomers of the RAD51 protein (a functional homolog of the Escherichia coli RecA recombinase) to 39-ending single-stranded DNA overhangs created by nucleolytic resection (Van Den Bosch et al. 2003). The resulting RAD51 nucleoprotein filament promotes formation of a joint molecule between the processed broken DNA and the homologous repair template by the orchestrated steps of homology searching and DNA strand invasion and exchange. Joint molecule formation is followed by new DNA synthesis, which replaces nucleotides lost through DSB formation and 39-end resection (Pâques and Haber 1999;Symington 2002;Li and Heyer 2008). Depending on the subpathway of HR, subsequent steps may involve unwinding (dissolution) of the DNA strand containing the newly synthesized DNA and ligation to the processed second end or the formation of a stably joined Holliday junction intermediate that can be processed further by structure-specific endonucleases yielding recombinant products (Li and Heyer 2008).At the heart of the HR process is the Rad51 (or RecA) nucleoprotein filament, whose formation requires the initial association of four to five monomers with 39-ending singlestranded DNA in the step known as nucleation (Galletto

Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3

“…Previous studies in humans suggested that the minimal efficient processing segment substrates of Rad51 were 300-500 bp with uninterrupted homology (60), which would preclude the use of Alu elements. More recent studies of Rad51 − or apparently Rad51-dependent processes, however, questioned these strict limits (61,62). Thus, Rad51 or another of its paralogs in humans (63) may indeed potentially mediate BIR using Alu elements as substrates, although less efficiently, also explaining the rarity of identical Alu-Alu-mediated CNVs.…”

Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filamentin vivo

“…Interestingly, the inhibitory effects of the Rad51 catalytic mutants are not equivalent; Rad51-K133A is considerably more toxic. Importantly, 3' polymerization in cell lines expressing Rad51-K133A 23 or Rad51-K133R was not restored following provision of an additional ~ 2-fold excess of ectopic wild-type FLAG-Rad51 (a level similar to that previously found to elevate 3' polymerization in the background of endogenous wild-type Rad51) 23 . Therefore, we are the first to show that the toxic effects of the Rad51 catalytic mutants on HR arise via dominant-negative inhibition of wild-type Rad51 function and occur at the fundamental level of the Rad51 nucleoprotein filament in vivo.…”

The Dichotomous Effects of Caffeine on Homologous Recombination in Mammalian Cells

“…Given current models for HR that posit 3' polymerization as an expected early intermediate step in the pathway of HR (78), we fully expected gene targeting, like 3' polymerization, to be enhanced by caffeine exposure. Also, our previous studies suggest a link between trends in 3' polymerization and gene targeting (26,29,33,51,79). Thus, we examined possible explanations for this dichotomy.…”