Expression of PCNA-binding domain of CtIP, a motif required for CtIP localization at DNA replication foci, causes DNA damage and activation of DNA damage checkpoint

Abstract

CtIP, CtBP-interacting protein, is a nuclear protein that was identified as a cofactor for the transcriptional repressor CtBP. Our genetic studies in mice revealed that haploid insufficiency of CtIP leads to tumorigenesis and is associated with shortened life span. At the molecular level, CtIP is a multivalent adaptor. It interacts directly with pRB family members, the prototype tumor suppressor proteins, and contributes to G1/S regulation. It has also been implicated in DNA damage checkpoint control through its interaction with the breast cancer susceptibility gene product BRCA1. Recently, it was found to modulate the nuclease activity of the Mre11/Rad50/NBS1 complex. Here we report that CtIP is recruited to S-phase DNA replication foci through a novel motif functioning as replication foci targeting sequence (RFTS). This motif contains a consensus PCNA-interacting protein box that binds to PCNA both in vivo and in vitro. In support of the biological significance of this interaction, we detected arrest of the cell cycle at the S/G2 phase transition, and suppression of cell proliferation in U2-OS cells upon the conditional expression of the wild type, but not a mutated RFTS using a tetracycline-inducible system. We found that cells expressing RFTS had excess DNA double strand breaks as demonstrated by formation of γ-H2AX nuclear foci. Finally, G2/M checkpoint activation in response to the expression of the CtIP RFTS is abrogated by caffeine treatment. Our work suggests an intimate relationship between CtIP and PCNA may be important for the maintenance of genomic stability in higher eukaryotic organism.