Abstract
Synthesis-dependent strand annealing (SDSA) is the preferred mode of homologous recombination in somatic cells leading to an obligatory non-crossover outcome, thus avoiding the potential for chromosomal rearrangements and loss of heterozygosity. Genetic analysis identified the Srs2 helicase as a prime candidate to promote SDSA. Here, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion and with a distinct polarity. Specifically, we partly reconstitute the SDSA pathway using Rad51, Rad54, RPA, RFC, DNA Polymerase δ with different forms of PCNA. Consistent with genetic data showing the requirement for SUMO and PCNA binding for the SDSA role of Srs2, Srs2 displays a slight but significant preference to disrupt extending D-loops over unextended D-loops when SUMOylated PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA. Our data establish a biochemical mechanism for the role of Srs2 in crossover suppression by promoting SDSA through disruption of extended D-loops.
Highlights
Loss of heterozygosity (LOH) can be an important contributor to carcinogenesis and a principal mechanism leading to LOH is crossover formation by homologous recombination (HR) (Knudson, 2001; Stern, 1936)
Consistent with the genetic data, we demonstrate that Srs2 disrupts D-loops in an ATP-dependent fashion with a slight but significant preference for extending D-loops over unextended D-loops when SUMO-PCNA is present, compared to unmodified PCNA or monoubiquitinated PCNA (Ubi-PCNA)
Short ssDNAs (~35–100 nts) are used for Rad51 filament formation and DNA strand invasion, the D loops products in such reactions suffer from instability due to Rad54’s ability to dismantle these short D loops efficiently (Bugreev et al, 2007b; Wright and Heyer, 2014)
Summary
Loss of heterozygosity (LOH) can be an important contributor to carcinogenesis and a principal mechanism leading to LOH is crossover formation by homologous recombination (HR) (Knudson, 2001; Stern, 1936). HR is a high-fidelity DNA repair pathway for DNA double-strand breaks (DSB) and interstrand cross-links (Heyer et al, 2010). Crossovers between non-allelic DNA regions lead to chromosomal aberrations including deletions, inversions, and translocations. Non-crossover is the preferred outcome of HR in somatic cells avoiding potential loss of heterozygosity and possible chromosome rearrangements. The D-loop is the joint molecule produced by the Rad51-DNA filament after homology search and DNA strand invasion, key steps of the HR process (Heyer et al, 2010).
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