Dynamics at the Fork: Molecular Dissection of Human DNA Replication and Fork Reversal Mechanisms

Faithful DNA replication is essential for genome integrity, yet the replication machinery, the replisome, is frequently challenged by replication stress. When unresolved, replication stress leads to genome instability, a hallmark of cancer and genetic disorders. Fork reversal is a critical stress response mechanism that stabilises stalled forks and enables fork restart, but how reversal factors coordinate with the replisome and engage stressed forks to promote fork reversal remains poorly defined. My project addresses these gaps by developing novel, integrated single-molecule and biochemical approaches to visualise human DNA replication and fork reversal dynamics at single-molecule resolution. These methods will allow me to track replisome composition, motion, and reversal factor recruitment in real time. I will systematically dissect replisome dynamics during normal replication, under stress, and how reversal factors access stressed forks to facilitate reversal in coordination with the replisome. Complementary AFM imaging will provide structural validation of reversed forks and replication intermediates, defining key architectural features across distinct stages of reversal. My work will uncover key mechanisms underlying human DNA replication, the replisome’s response to stress and fork reversal. It will also establish a robust platform for investigating diverse replication-associated processes.