Rad52/Rad59-dependent recombination as a means to rectify faulty okazaki fragment processing

Abstract

Correct removal of 5’-flap structures formed by Pol $\delta$ for the maturation of Okazaki fragments during lagging strand DNA synthesis is crucial for stable maintenance of genetic materials and cell viability. In this study, we identified RAD52 as a multi-copy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. Rad52 is a key recombination protein that contains recombination mediator and DNA annealing activities, playing an essential role for general homologous recombination. In contrast, overexpression of Rad51 that works conjointly with Rad52 failed to suppress the dna2-K1080E mutation, raising the possibility that Rad52 plays a unique role in facilitating processing of Okazaki fragment processing. In keeping with this notion, the recombination mediator, i.e., Rad51-binding activity of Rad52 was dispensable for the suppression of dna2-K1080E, whereas both the DNA annealing activity and the ability of Rad52 to interact with Rad59 were required. Furthermore, Rad52-QDDD/AAAA, a mutant Rad52 that is defective in binding replication protein-A did not rescue the dna2-K1080E mutation. We also found that both Rad52 and Rad52-QDDD/AAAA proteins were able to stimulate endonuclease activities of Dna2 and Rad27, indicating that stimulation of Dna2 or Rad27 by Rad52 is not a direct cause of suppression observed. In addition, Elg1 and Rsc2, required for sister-chromatid cohesion was found to be essential for the Rad52-dependent suppression of dna2-K1080E, suggesting that the suppression occurred via Rad51-independent sister-chromatid recombination event. Our findings suggest a novel Rad52-dependent, but Rad51-independent recombination pathway that could ultimately lead to removal of flaps that have escaped endonucleolytic cleavage by Dna2.