The Replication Stress Response to Selective Stalling of the Leading and Lagging Strands

建议书摘要 目前在DNA修复和应激反应方面的研究通过拖延两者来询问未知的问题 DNA链(领先和落后)。然而，复制过程在每条链上发生的方式不同，并且 在细菌等生物体中的证据表明，当领先或落后的链 都被明确地搁置了。这项建议将研究复制应激反应的差异 障碍和阻碍首次被引入到人类细胞的领先和落后链中。vbl.使用 多种新颖的方法，该提案旨在专门阻止每条复制的DNA链。在选择之后 STALING I将评估复制分叉进展率，验证链特定的STARTING，并表征 被招募到复制叉子上的蛋白质，同时回答关于叉子颠倒的问题，在失速后重新启动， 以及发信号通知先前不能询问的事件。尽管我预计复制过程中会有所不同 压力反应当比较领先和滞后的失速时，任何结果都将是第一个 描述人类细胞中的链特异性停滞，既代表挑战又代表机遇。 最终，这项提议将推进DNA复制应激反应领域，建立方法来 询问更多分裂细胞每天遇到的生理障碍。

PROPOSAL SUMMARY Current studies in DNA repair and the stress response interrogate unknown questions by stalling both DNA strands (leading and lagging). However, the process of replication occurs differently on each strand, and evidence in organisms such as bacteria demonstrate different responses when the leading or lagging strands are specifically stalled. This proposal will investigate differences in the replication stress response when obstacles and stalls are introduced into the leading and lagging strands in human cells for the first time. Using multiple novel approaches, the proposal aims to specifically stall each replicating DNA strand. After selective stalling I will assess replication fork progression rate, validate strand specific stalling, and characterize the proteins recruited to the replication fork, while answering questions about fork reversal, repriming after a stall, and signaling events that could not be previously interrogated. Although I expect differences in the replication stress response when comparing a leading and lagging strand stall, any result would be the first characterization of a strand-specific stall in human cells, representing both a challenge and opportunity. Ultimately, this proposal will advance the DNA replication stress response field, establishing methods to interrogate more physiological obstacles that dividing cells encounter daily.