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.
The replication stress response has evolved to repair or bypass lesions within DNA and is critical to prevent genetic instability and malignancy. Although there is a small body of work that suggests a differential replication stress response that mounts on the leading strand versus the lagging strand in organisms such as E coli, to date, there is no evidence that this is the case in humans as all past approaches have assumed a holistic view of the replication fork. This proposal aims to move the DNA repair, cancer biology, and replication stress response fields forward by developing multiple approaches to interrogate the replication stress response specifically when the leading or lagging DNA strands encounter an obstacle or lesion.
