CTF18 interacts with replication protein A in response to replication stress

Abstract

Replication stress response is a protective mechanism against defects in chromosome replication for maintaining genome integrity in eukaryotic cells. An alternative clamp loader complex termed chromosome transmission fidelity protein 18 and replication factor C (CTF18-RFC) has been shown to act as a positive regulator of two types of replication stress response: S-phase checkpoint signaling and translesion DNA synthesis. However, it remains largely unknown how CTF18-RFC responds to replication stress and is recruited to stalled replication forks. The present study demonstrated that endogenous CTF18 forms a physical complex with a single-stranded DNA-binding protein replication protein A (RPA) in mammalian cells. Using an in situ proximity ligation assay (PLA), it was demonstrated that the interaction between CTF18 and RPA occurs in chromatin when replication stress is elicited by treatment with hydroxyurea during S phase. Similar results were obtained after exposure to ultraviolet irradiation, which triggers translesion DNA synthesis. Furthermore, the PLA demonstrated that the kinetics of the interaction between CTF18 and RPA was positively correlated with that of checkpoint kinase 1 phosphorylation, which is an indicator of activation of the ATM and Rad3-related pathway. These findings provide novel insights into the molecular mechanism underlying the participation of CTF18-RFC in the regulation of replication stress response.