Dual functions of DNA replication forks in checkpoint signaling and PCNA ubiquitination

Abstract

During cell proliferation, DNA damage inflicted by intrinsic or extrinsic genotoxic stresses impose a threat to DNA replication. The stability of the DNA replication forks that encounter DNA damage is crucial for genomic integrity. Both the ATR-regulated checkpoint pathway and the translesion DNA synthesis mediated by the ubiquitinated PCNA are important for continuous replication of damaged DNA. We have recently shown that Chk1, a key effector kinase of ATR in checkpoint response, is required for efficient PCNA ubiquitination after DNA damage. Surprisingly, the ubiquitination of PCNA is independent of ATR, but regulated by Claspin, a replication protein that mediates the activation of Chk1 by ATR. Like Claspin, Timeless and Rad17, two other Chk1 regulators at stressed replication forks, are also implicated in PCNA ubiquitination. These findings suggest that while ATR signaling and PCNA ubiquitination are two independent processes, they are mediated by a common group of proteins including Chk1 and it regulators at replication forks. Furthermore, these data raise the possibility that Chk1 and its regulators may constitute a functional module at replication forks to enable multiple stress responses.

Figure 1

Regulation of PCNA ubiquitination by Usp1 and Rad17. (A) Depletion of Usp1 leads to elevated levels of ubiquitinated PCNA in undamaged and damaged cells. HeLa cells transfected with Usp1 siRNA or control siRNA were treated with UV or left untreated. The unmodified and ubiquitinated PCNA was detected with an anti-PCNA antibody. (B) Depletion of Rad17 leads to reduced levels of ubiquitinated PCNA in UV- and HU-treated cells. Cells transfected with Rad17 siRNA or control siRNA were treated with UV and HU as indicated, and were analyzed by immunoblotting as above.

Figure 2

A model for a multi-functional module at DNA replication forks. Chk1, Claspin, Timeless, and Tipin may constitute a functional module at replication forks. Through its interactions with RPA-ssDNA, 9–1–1, and PCNA, this module may play dual roles in ATR checkpoint signaling and PCNA ubiquitination.