Understanding replisome dynamics by proteomics and functional genomics

Abstract

The maintenance of genome integrity is critical for the survival of organisms as it ensures the faithful duplication of genetic material and passage to daughter cells during cell division. DNA replication is an integral process in maintaining genome integrity and requires precise orchestration of replisome components. However, endogenous and exogenous sources of replication stress constantly challenge this process. Cells are equipped with surveillance mechanisms to detect and elicit corresponding responses in the face of these obstacles. Defects in any of these mechanisms increase the susceptibility of DNA to mutate and the likelihood of structural alterations of chromosomes, leading to genomic instability, a hallmark of cancers. A comprehensive study of DNA replication and the replication stress response will advance our understanding of the cellular networks that maintain genome integrity and provide insight into developing novel therapeutic strategies. In this dissertation, I utilized omics-based approaches, including proteomics and functional genetic screens, to establish a comprehensive picture of DNA replication. In chapter 2, we aimed to improve the existing replisome retrieval technique – the isolation of proteins on nascent DNA (iPOND). iPOND generally has high contamination of post-replicative material and is biased toward identifying highly abundant proteins when combined with labeled mass spectrometry (MS). We developed a method named iPOND2-DRIPPER by combining density-based replisome enrichment and label-free MS. iPOND2-DRIPPER resulted in 5-10-fold increased replisome yield over previously reported methods. With substantially improved enrichment, iPOND2-DRIPPER allowed direct quantification of ubiquitination events at active or stalled replication forks. Additionally, iPOND2-DRIPPER uncovered the spatial-temporal association of stalled replication forks with the nuclear periphery. Chapter 3 focused on ring finger protein 4 (RNF4), one of two SUMO-targeted E3 ubiquitin ligases (STUbLs) in mammalian cells. RNF4 is involved in several genome maintenance pathways including double-strand break (DSB) repair and the resolution of DNA-protein crosslinks (DPCs). However, its role in DNA replication has remained obscure. We utilized clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) genetic screens and uncovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase (USP7) for survival in noncancerous cells. We demonstrated that RNF4 and USP7 cooperatively contribute to sustaining DNA replication. Their cooperation is particularly important when the proteasome is inhibited by bortezomib, which restrains the nuclear ubiquitin pool. We showed that upon proteasome inhibition, RNF4-/-/USP7-/- mutants exhibited substantially dysregulated DNA replication and compromised checkpoint response, resulting in elevated cell death. These findings suggest that RNF4 and USP7 work in parallel to modulate the nuclear ubiquitin pool required for genome maintenance. In conclusion, this dissertation has generated valuable datasets that will not only expand our understanding of DNA replication but also provide insights for future investigations into genome maintenance mechanisms.