Reconstitution of Human Claspin-mediated Phosphorylation of Chk1 by the ATR (Ataxia Telangiectasia-mutated and Rad3-related) Checkpoint Kinase

Laura A Lindsey-Boltz,Özdemirhan Serçin,Jun-Hyuk Choi,Aziz Sancar

doi:10.1074/jbc.m109.064485

Abstract

ATR (ATM and Rad3-related) initiates a DNA damage signaling pathway in human cells upon DNA damage induced by UV and UV-mimetic agents and in response to inhibition of DNA replication. Genetic data with human cells and in vitro data with Xenopus egg extracts have led to the conclusion that the kinase activity of ATR toward the signal-transducing kinase Chk1 depends on the mediator protein Claspin. Here we have reconstituted a Claspin-mediated checkpoint system with purified human proteins. We find that the ATR-dependent phosphorylation of Chk1, but not p53, is strongly stimulated by Claspin. Similarly, DNA containing bulky base adducts stimulates ATR kinase activity, and Claspin acts synergistically with damaged DNA to increase phosphorylation of Chk1 by ATR. Mutations in putative phosphorylation sites in the Chk1-binding domain of Claspin abolish its ability to mediate ATR phosphorylation of Chk1. We also find that a fragment of Claspin containing the Chk1-binding domain together with a domain conserved in the yeast Mrc1 orthologs of Claspin is sufficient for its mediator activity. This in vitro system recapitulates essential components of the genetically defined ATR-signaling pathway.

Highlights

57-amino acid minimal Chk1-binding domain (CKBD) of Xenopus Claspin [8]
We find that Claspin strongly stimulates the TopBP1-dependent ATR phosphorylation of Chk1 but not phosphorylation of other ATR substrates such as p53, and this stimulation is amplified by damaged DNA
Claspin Stimulates ATR Kinase Activity Toward Chk1—It has been reported that ATR phosphorylation of Chk1 on Ser345, but not phosphorylation of other ATR substrates, such as p53, is dependent on Claspin in vivo [6]

Summary

Introduction

57-amino acid minimal Chk1-binding domain (CKBD) of Xenopus Claspin [8]. Mutations in this domain abrogate Claspin-Chk association as well as the phosphorylation of Chk by ATR in both Xenopus (8 –10) and human systems [11, 12]. Genetic studies in human cell lines and in vitro studies with HeLa cell-free extracts and Xenopus egg extracts have identified many proteins including RPA, the ATR-ATRIP heterodimer, the Rad17-RFC1⁄79-1-1 and Timeless1⁄7Tipin checkpoint complexes, TopBP1, and Claspin as essential components for phosphorylation of Chk and activating the DNA damage or replication checkpoints [1]. The ATR 3 Chk signaling pathway in these in vitro studies was not dependent on the other checkpoint proteins known to be required for ATR activation. Tel.: 919-962-0115; E-mail: Aziz_Sancar@ med.unc.edu. 2 The abbreviations used are: ATM, ataxia telangiectasia-mutated; ATR, ATM and Rad3-related; ATRIP, ATR-interacting protein; CKBD, Chk1-binding domain; BPDE, Benzo[a]pyrene diol epoxide; GST, glutathione S-transferase; kd, kinase dead; RPA, replication protein A

Results

Discussion

Conclusion