Saccharomyces cerevisiae DNA polymerase IV overcomes Rad51 inhibition of DNA polymerase δ in Rad52-mediated direct-repeat recombination

• Published in: Nucleic acids research Vol. 51; no. 11; pp. 5547 - 5564
• Main Authors: Cerqueira, Paula G, Meyer, Damon, Zhang, Lilin, Mallory, Benjamin, Liu, Jie, Hua Fu, Becky Xu, Zhang, Xiaoping, Heyer, Wolf-Dietrich
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 23.06.2023
• Subjects: DNA - metabolism; DNA Polymerase beta - genetics; DNA Polymerase III - genetics; DNA Polymerase III - metabolism; DNA Repair; Genome Integrity, Repair and; Humans; Rad51 Recombinase - genetics; Rad51 Recombinase - metabolism; Rad52 DNA Repair and Recombination Protein - genetics; Rad52 DNA Repair and Recombination Protein - metabolism; Recombinational DNA Repair; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkad281

Abstract Saccharomyces cerevisiae DNA polymerase IV (Pol4) like its homolog, human DNA polymerase lambda (Polλ), is involved in Non-Homologous End-Joining and Microhomology-Mediated Repair. Using genetic analysis, we identified an additional role of Pol4 also in homology-directed DNA repair, specifically in Rad52-dependent/Rad51-independent direct-repeat recombination. Our results reveal that the requirement for Pol4 in repeat recombination was suppressed by the absence of Rad51, suggesting that Pol4 counteracts the Rad51 inhibition of Rad52-mediated repeat recombination events. Using purified proteins and model substrates, we reconstituted in vitro reactions emulating DNA synthesis during direct-repeat recombination and show that Rad51 directly inhibits Polδ DNA synthesis. Interestingly, although Pol4 was not capable of performing extensive DNA synthesis by itself, it aided Polδ in overcoming the DNA synthesis inhibition by Rad51. In addition, Pol4 dependency and stimulation of Polδ DNA synthesis in the presence of Rad51 occurred in reactions containing Rad52 and RPA where DNA strand-annealing was necessary. Mechanistically, yeast Pol4 displaces Rad51 from ssDNA independent of DNA synthesis. Together our in vitro and in vivo data suggest that Rad51 suppresses Rad52-dependent/Rad51-independent direct-repeat recombination by binding to the primer-template and that Rad51 removal by Pol4 is critical for strand-annealing dependent DNA synthesis. Graphical Abstract Graphical Abstract Rad51 inhibits DNA synthesis by Pold (use Greek delta for last letter) during Rad52-mediated repeat recombination. Poll (use Greek lambda for last letter) can displace Rad51 to allow PCNA loading and Pold (use Greel for last letter) DNA synthesis.