Center for Cell Structure and Function, Shandong Provincial Key Laboratory of Animal Resistance Biology, College of Life Sciences, Shandong Normal University, Jinan, Shandong 250014, China.
Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250012, China.
Nucleic Acids Res. 2023 Aug 25;51(15):7914-7935. doi: 10.1093/nar/gkad537.
During the repair of DNA double-strand breaks (DSBs), de novo synthesized DNA strands can displace the parental strand to generate single-strand DNAs (ssDNAs). Many programmed DSBs and thus many ssDNAs occur during meiosis. However, it is unclear how these ssDNAs are removed for the complete repair of meiotic DSBs. Here, we show that meiosis-specific depletion of Dna2 (dna2-md) results in an abundant accumulation of RPA and an expansion of RPA from DSBs to broader regions in Saccharomyces cerevisiae. As a result, DSB repair is defective and spores are inviable, although the levels of crossovers/non-crossovers seem to be unaffected. Furthermore, Dna2 induction at pachytene is highly effective in removing accumulated RPA and restoring spore viability. Moreover, the depletion of Pif1, an activator of polymerase δ required for meiotic recombination-associated DNA synthesis, and Pif1 inhibitor Mlh2 decreases and increases RPA accumulation in dna2-md, respectively. In addition, blocking DNA synthesis during meiotic recombination dramatically decreases RPA accumulation in dna2-md. Together, our findings show that meiotic DSB repair requires Dna2 to remove ssDNA-RPA filaments generated from meiotic recombination-associated DNA synthesis. Additionally, we showed that Dna2 also regulates DSB-independent RPA distribution.
在 DNA 双链断裂 (DSB) 的修复过程中，新合成的 DNA 链可以取代亲本链，产生单链 DNA (ssDNA)。减数分裂过程中会发生许多程序性 DSB，因此会产生许多 ssDNA。然而，这些 ssDNA 如何被清除以完成减数分裂 DSB 的修复尚不清楚。在这里，我们表明，减数分裂特异性耗尽 Dna2 (dna2-md) 会导致 RPA 的大量积累，并将 RPA 从 DSB 扩展到酿酒酵母中更广泛的区域。结果，DSB 修复出现缺陷，孢子无法存活，尽管交叉/非交叉的水平似乎不受影响。此外，在粗线期诱导 Dna2 对于去除积累的 RPA 和恢复孢子活力非常有效。此外，聚合酶 δ 的激活因子 Pif1 的缺失，以及减数分裂重组相关 DNA 合成所必需的 Pif1 抑制剂 Mlh2，分别减少和增加了 dna2-md 中的 RPA 积累。此外，在减数分裂重组过程中阻断 DNA 合成会显著减少 dna2-md 中的 RPA 积累。总之，我们的研究结果表明，减数分裂 DSB 修复需要 Dna2 来去除与减数分裂重组相关的 DNA 合成产生的 ssDNA-RPA 纤维。此外，我们还表明，Dna2 还调节 DSB 非依赖性 RPA 分布。
