Chromatin remodeler Fun30 promotes efficient DSB end resection and recombination during yeast meiosis

Resection, nucleolytic processing of DSB ends is necessary to generate 3' single-stranded DNA tails (ssDNA) for homologous recombination (HR). Meiotic recombination initiated by Spo11 induced double-strand breaks (DSBs) is essential for the accurate segregation of homologous chromosomes. After cleavage, Spo11 stays covalently linked to the DSB ends, which requires MRX/Sae2 incision on broken molecules to allow following Exo1-mediated resection. Exonuclease I activity is inhibited by nucleosome bound DNA in vitro. To ensure resection proceeds, resection machineries must overcome chromatin barrier. Here we show that in the absence of Fun30, an SNF2-like ATPase, resection tract lengths shortened, albeit less severe than exo1-nd (with more longer resection tracts than that in exo1nd), across all DSB hotspots, suggesting that Fun30 is required for meiotic resection. Additive resection defect in fun30Δ exo1-nd (nuclease dead) compared to exo1-nd mutant indicates that Fun30 regulates MRX/Sae2 nicking positions. We also observed extremely short resection tracts in the double mutants are mostly confined to NDR, suggesting initial nicking step is blocked by nucleosomes.双链断裂（double-strand break, DSB）末端的核酸酶解加工与切除过程，是生成用于同源重组（homologous recombination, HR）的3'单链DNA尾（ssDNA）的必要前提。由Spo11诱导产生的双链断裂所启动的减数分裂重组，对于同源染色体的精准分离至关重要。切割完成后，Spo11会与DSB末端共价结合，这一过程需要断裂分子上的MRX/Sae2复合物进行切口切割，才能允许后续由外切酶1（Exonuclease I, Exo1）介导的末端切除。体外实验表明，外切酶I的活性会被结合有核小体的DNA所抑制。为保障末端切除顺利推进，切除相关分子机器必须突破染色质屏障。本研究显示，在类SNF2 ATP酶（SNF2-like ATPase）Fun30缺失的情况下，所有DSB热点区域的切除片段长度均出现缩短；尽管其缺陷程度相较于exo1-nd突变体更温和，且exo1-nd突变体的长切除片段占比更高，这表明Fun30是减数分裂末端切除所必需的因子。相较于单一exo1-nd突变体，fun30Δ exo1-nd（核酸酶失活型）双突变体的末端切除缺陷呈现累加效应，这表明Fun30可调控MRX/Sae2的切口位点。我们还观察到，双突变体中极短的切除片段大多局限于核小体缺失区域（nucleosome-depleted region, NDR），这提示初始切口步骤会被核小体所阻断。