Department of Microbiology and Molecular Genetics, University of California, Davis, One Shields Avenue, Davis, CA 95616-8665, USA.
Department of Molecular and Cellular Biology, University of California, Davis, One Shields Avenue, Davis, CA 95616-8665, USA.
Nucleic Acids Res. 2023 Jun 23;51(11):5547-5564. doi: 10.1093/nar/gkad281.
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.
酿酒酵母 DNA 聚合酶 IV（Pol4）与其同源物人类 DNA 聚合酶 λ（Polλ）一样，参与非同源末端连接和微同源介导的修复。通过遗传分析，我们发现 Pol4 还有一个额外的作用，即在同源定向 DNA 修复中，特别是在 Rad52 依赖性/ Rad51 非依赖性直接重复重组中。我们的结果表明，Pol4 在重复重组中的需求被 Rad51 的缺失所抑制，这表明 Pol4 拮抗 Rad51 对 Rad52 介导的重复重组事件的抑制。使用纯化的蛋白质和模型底物，我们在体外反应中重新模拟了直接重复重组过程中的 DNA 合成，结果表明 Rad51 直接抑制 Polδ DNA 合成。有趣的是，尽管 Pol4 本身不能进行广泛的 DNA 合成，但它有助于 Polδ 克服 Rad51 对 DNA 合成的抑制。此外，在含有 Rad52 和 RPA 的反应中，当 DNA 链退火是必需的时，Pol4 依赖性和 Rad51 存在时 Polδ DNA 合成的刺激都发生在 Pol4 中。从机制上讲，酵母 Pol4 可以在不依赖于 DNA 合成的情况下将 Rad51 从 ssDNA 上置换下来。我们的体内和体外数据表明，Rad51 通过结合引物模板来抑制 Rad52 依赖性/ Rad51 非依赖性直接重复重组，而 Rad51 由 Pol4 去除是链退火依赖性 DNA 合成的关键。
