The role of replisome degradation in replication fork collapse and DSB formation

描述(申请人提供)：癌症最常见的原因之一是双链断裂(DSB)的积累，这在DNA复制过程中经常发生。当DNA复制被阻止时，复制的解旋酶活性从新合成的子链上解偶联，导致单链DNA的很长一段。ATR-CHK1检查点通路识别这些结构，稳定这些停滞不前的分叉，并抑制细胞周期进展。ATR的丢失允许将停滞的复制分叉处理为反向分叉和DSB，从而防止重新启动复制。目前尚不清楚在没有ATR的情况下，停滞不前的复制叉子如何成为DSB。我们最近的数据表明，PLK1和RNF4，一种针对相扑的泛素E3连接酶，在复制分叉停滞的位置驱动复制体的降解。我假设，依赖PLK1和RNF4的泛素化和复制体组分的移除是G2-M转变中自然发生的过程，当ATR缺失时，这一过程在停滞的叉子上过早发生。此外，我认为这种去除刺激了SLX4-核酸内切酶复合体的招募，该复合体直接介导复制分叉停滞部位DSB的形成。为了测试我的模型，我将量化在细胞周期的不同阶段以及在没有ATR的情况下复制叉停滞期间染色质上复制体组分的水平。此外，我将开发新的系统来测试叉子加工和DSB形成是否需要去除复制体成分。这些实验将扩大我们对复制分叉崩溃和维持基因组完整性的理解。通过这样做，我们对癌症病因学和治疗的知识将得到扩展。

DESCRIPTION (provided by applicant): One of the most common causes of cancer is accumulation of double-stranded breaks (DSBs), which occurs frequently during DNA replication. When DNA replication is blocked, replicative helicase activity becomes uncoupled from the newly synthesized daughter strand, resulting in long stretches of single-stranded DNA. The ATR-CHK1 checkpoint pathway recognizes these structures and stabilizes these stalled forks and inhibits cell cycle progression. Loss of ATR allows stalled replication forks to be processed into reversed forks and DSBs, which prevents the re-initiation of replication. It is unclear how stalled replication forks become DSBs in the absence of ATR. Our recent data suggests that PLK1 and RNF4, a SUMO-targeted ubiquitin E3 ligase, drive replisome degradation at sites of replication fork stalling. I hypothesize that PLK1- and RNF4-dependent ubiquitination and removal of replisome components is a naturally occurring process at the G2-M transition which happens prematurely at stalled forks when ATR is absent. Furthermore, I propose that this removal stimulates the recruitment of the SLX4- endonuclease complex, which directly mediates DSB formation at sites of replication fork stalling. To test my models, I will quantify levels of replisome components on chromatin at different phases of the cell cycle and during replication fork stalling in the absence of ATR. In addition, I will develop novel systems t test whether the removal of replisome components is required for fork processing and DSB formation. These experiments will expand our understanding of replication fork collapse and the maintenance of genome integrity. In so doing, our knowledge of cancer etiology and treatment will be extended.