# Replisome Proximal Protein Associations and Dynamic Proteomic Changes at Stalled Replication Forks

## TL;DR

This study maps protein interactions at DNA replication forks and shows how they change under stress, offering new insights into DNA replication and repair.

## Contribution

A comprehensive proteomic framework of replisome protein associations under normal and stressed conditions is presented.

## Key findings

- Hydroxyurea treatment caused 108 proteomic changes, with 45 enriched and 63 depleted associations.
- The replisome undergoes dynamic reorganization under genotoxic stress.
- New putative players in DNA replication arrest response were identified.

## Abstract

DNA replication is a fundamental cellular process that ensures the transfer of genetic information during cell division. Genome duplication takes place in S phase and requires a dynamic and highly coordinated recruitment of multiple proteins at replication forks. Various genotoxic stressors lead to fork instability and collapse, hence the need for DNA repair pathways. By identifying the multitude of protein interactions implicated in those events, we can better grasp the complex and dynamic molecular mechanisms that facilitate DNA replication and repair. Proximity-dependent biotin identification was used to identify associations with 17 proteins within four core replication components, namely the CDC45/MCM2-7/GINS helicase that unwinds DNA, the DNA polymerases, replication protein A subunits, and histone chaperones needed to disassemble and reassemble chromatin. We further investigated the impact of genotoxic stress on these interactions. This analysis revealed a vast proximity association network with 108 nuclear proteins further modulated in the presence of hydroxyurea; 45 being enriched and 63 depleted. Interestingly, hydroxyurea treatment also caused a redistribution of associations with 11 interactors, meaning that the replisome is dynamically reorganized when stressed. The analysis identified several poorly characterized proteins, thereby uncovering new putative players in the cellular response to DNA replication arrest. It also provides a new comprehensive proteomic framework to understand how cells respond to obstacles during DNA replication.

•Protein associations for 17 replication fork components are provided as a resource.•Identification of proteomic changes due to hydroxyurea-induced replicative stress.•Molecular insights on fork dynamics and DNA replication repair modulators.

Protein associations for 17 replication fork components are provided as a resource.

Identification of proteomic changes due to hydroxyurea-induced replicative stress.

Molecular insights on fork dynamics and DNA replication repair modulators.

DNA replication repair was examined by comparing proximal protein associations for 17 core components of the replication fork in cycling cells and cells facing genotoxic stress. An important reorganization of the interactome was observed upon a hydroxyurea treatment, with 108 associations being either markedly lost or gained. This high-resolution analysis informs on replication fork protein complexes, organization, and stress responses. Our rich data resource includes interactome data for components of the CDC45/MCM2-7/GINS (CMG) helicase, histone chaperones, and DNA polymerases.