Interaction of yeast Rad51 and Rad52 relieves Rad52-mediated inhibition of <i>de novo</i> telomere addition

DNA double-strand breaks (DSBs) are toxic forms of DNA damage that must be repaired to maintain genome integrity. Telomerase can act upon a DSB to create a de novo telomere, a process that interferes with normal repair and creates terminal deletions. We previously identified sequences in Saccharomyces cerevisiae (SiRTAs; Sites of Repair-associated Telomere Addition) that undergo unusually high frequencies of de novo telomere addition, even when the original chromosome break is several kilobases distal to the eventual site of telomerase action. Association of the single-stranded telomere binding protein Cdc13 with a SiRTA is required to stimulate de novo telomere addition. Because extensive resection must occur prior to Cdc13 binding, we utilized these sites to monitor the effect of proteins involved in homologous recombination. We find that telomere addition is significantly reduced in the absence of the Rad51 recombinase, while loss of Rad52, required for Rad51 nucleoprotein filament formation, has no effect. Deletion of RAD52 suppresses the defect of the rad51Δ strain, suggesting that Rad52 inhibits de novo telomere addition in the absence of Rad51. The ability of Rad51 to counteract this effect of Rad52 does not require DNA binding by Rad51, but does require interaction between the two proteins, while the inhibitory effect of Rad52 depends on its interaction with Replication Protein A (RPA). Intriguingly, the genetic interactions we report between RAD51 and RAD52 are similar to those previously observed in the context of checkpoint adaptation. Forced recruitment of Cdc13 fully restores telomere addition in the absence of Rad51, suggesting that Rad52, through its interaction with RPA-coated single-stranded DNA, inhibits the ability of Cdc13 to bind and stimulate telomere addition. Loss of the Rad51-Rad52 interaction also stimulates a subset of Rad52-dependent microhomology-mediated repair (MHMR) events, consistent with the known ability of Rad51 to prevent single-strand annealing.

DNA双链断裂（DNA double-strand breaks, DSBs）是一类具有毒性的DNA损伤类型，必须通过修复以维持基因组完整性。端粒酶可作用于DNA双链断裂位点以从头合成端粒，该过程会干扰正常修复通路并产生末端缺失。我们此前在酿酒酵母（Saccharomyces cerevisiae）中鉴定出一类序列（SiRTAs：修复相关端粒添加位点，Sites of Repair-associated Telomere Addition），这类序列会以异常高的频率发生从头端粒添加，即便原始染色体断裂位点距离最终端粒酶作用位点尚有数千碱基之遥。
单链端粒结合蛋白Cdc13与SiRTA的结合，是刺激从头端粒添加所必需的条件。由于Cdc13结合前必须发生广泛的DNA末端切除，我们利用这类位点来监测参与同源重组的蛋白质的调控效应。
我们发现，缺失重组酶Rad51时，端粒添加效率会显著降低；而Rad52的缺失——Rad52是Rad51核蛋白丝形成所必需的因子——则无此影响。敲除RAD52可逆转rad51Δ突变株的修复缺陷，这表明在Rad51缺失的情况下，Rad52会抑制从头端粒添加过程。Rad51抵消Rad52这一抑制作用的能力，并不依赖于Rad51自身的DNA结合活性，而是依赖于二者之间的蛋白质相互作用；而Rad52的抑制效应则取决于其与复制蛋白A（RPA, Replication Protein A）的相互作用。
有趣的是，我们所报道的RAD51与RAD52之间的遗传互作模式，与此前在检查点适应（checkpoint adaptation）语境下观察到的结果高度相似。强制招募Cdc13可完全恢复Rad51缺失株中的端粒添加效率，这表明Rad52通过与被RPA包被的单链DNA结合，抑制了Cdc13结合并刺激端粒添加的能力。破坏Rad51与Rad52的相互作用还会激活一类依赖Rad52的微同源介导修复（MHMR, microhomology-mediated repair）事件，这与已知的Rad51可抑制单链退火（single-strand annealing）的功能相符。