Determinants of post-replicative gap formation and filling during DNA replication stress response – DetGap

DNA replication is constantly challenged by obstacles such as DNA damage that can block its progression, leading to so-called “replication stress”. Replication stress poses a threat to genome stability and cell viability, and has been associated to human diseases. Mechanisms of DNA damage tolerance can counteract replication stress. One such mechanism involves replication fork restart downstream of the blocking damage through the synthesis of a de novo DNA primer (repriming) by the DNA primase and polymerase PRIMPOL. Repriming ensures replication fork progression but results in post-replicative single-stranded DNA gaps. These gaps are subsequently filled by gap-filling mechanisms such as translesion DNA synthesis and template switching. Despite growing interest in post-replicative gaps formed in response to replication stress, the molecular bases of the repriming mechanism – from gap formation to filling – remain elusive. In particular, the triggers of repriming at blocked replication forks as well as the molecular determinants of gap-filling pathway “choice” are still poorly understood. In this project, we aim to tackle these fundamental questions. Specifically, based on preliminary data, we seek to address the impact of (1) DNA damage load and type of DNA damage, (2) DNA damage response factors and (3) chromatin state on gap formation and filling. To maximize the chances of success of these aims, we have established a consortium of experts in DNA replication, DNA damage response and chromatin. Together, we will employ interdisciplinary strategies and integrate cutting-edge single-cell and single-molecule microscopy, proteomics and genome-wide approaches. Completing this project is critical for understanding how cells respond to replication stress and deciphering the intricate interplay between replication stress responses and chromatin.