The Interaction between Checkpoint Kinase 1 (Chk1) and the Minichromosome Maintenance (MCM) Complex Is Required for DNA Damage-induced Chk1 Phosphorylation

Xiangzi Han,Aaron Aslanian,Kang Fu,Toshiya Tsuji,Youwei Zhang

doi:10.1074/jbc.m114.575035

Abstract

Chk1 is an essential mediator of the DNA damage response and cell cycle checkpoint. However, how exactly Chk1 transduces the checkpoint signaling is not fully understood. Here we report the identification of the heterohexamic minichromosome maintenance (MCM) complex that interacts with Chk1 by mass spectrometry. The interaction between Chk1 and the MCM complex was reduced by DNA damage treatment. We show that the MCM complex, at least partially, contributes to the chromatin association of Chk1, allowing for immediate phosphorylation of Chk1 by ataxia telangiectasia mutated and Rad3-related (ATR) in the presence of DNA damage. Further, phosphorylation of Chk1 at ATR sites reduces the interaction between Chk1 and the MCM complex, facilitating chromatin release of phosphorylated Chk1, a critical step in the initiation and amplification of cell cycle checkpoint. Together, these data provide novel insights into the activation of Chk1 in response to DNA damage.

Highlights

Chk1 is a key mediator protein that regulates replication checkpoint
Chk1 Interacts with the minichromosome maintenance (MCM) Complex—Recent studies demonstrated that Chk1 resides both in the nucleus and in the cytoplasm [6, 19]
The results showed that the presence of ethidium bromide (EtBr) during Co-IP did not affect the interaction between Chk1 and MCM7 (Fig. 2A), supporting the notion that Chk1 interacts with MCM proteins independently of DNA

Summary

Background

Chk is a key mediator protein that regulates replication checkpoint. Results: Chk interacts with the MCM complex. Recent studies suggest a spatiotemporal regulation model of Chk, in which DNA damage induces ATR-dependent phosphorylation of Chk on chromatin followed by a rapid release of phospho-Chk from chromatin into soluble nucleus and the cytoplasm, where Chk activates the cell cycle checkpoints, as well as being degraded; the latter functions as an autoinhibitory mechanism to terminate the checkpoint signaling [6]. In this regard, chromatin association of Chk is crucial for checkpoint initiation. Chk phosphorylation at ATR sites reduces the interaction between Chk and the MCM complex, facilitating chromatin release of phospho-Chk, which likely will contribute to subsequent checkpoint activation

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION