PDF(Pubmed)
Abstract：
Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.
摘要：
同源性依赖性靶向DNA整合，通常被称为基因靶向，为精确的基因组修饰提供了强大的工具；然而，其基本机制在人类细胞中仍然知之甚少。在这里，我们揭示了一种不依赖于同源重组（HR）蛋白Rad51的非规范基因靶向机制。这种机制被Rad52抑制抑制，提示单链退火（SSA）的参与。当通过HR或末端连接途径进行的DSB修复有缺陷并且不需要等基因DNA时，SSA介导的基因靶向变得突出。允许5%的序列差异。有趣的是，Msh2损失，BLM损失，和诱导靶位点DNA断裂均显着并协同增强SSA介导的靶向整合。最值得注意的是,SSA介导的整合与细胞周期无关，也发生在G1期。我们的发现为Rad51独立的靶向整合提供了明确的证据，并揭示了调节SSA介导的靶向以及随机整合的多种机制。
