Related Experiment Video
Updated: Mar 22, 2026
Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
Published on: November 5, 2012
Conformational Change of Human Checkpoint Kinase 1 (Chk1) Induced by DNA Damage
Xiangzi Han1, Jinshan Tang2, Jingna Wang1
1From the Department of Pharmacology, Case Comprehensive Cancer Center, and.
Abstract:
Phosphorylation of Chk1 by ataxia telangiectasia-mutated and Rad3-related (ATR) is critical for checkpoint activation upon DNA damage. However, how phosphorylation activates Chk1 remains unclear. Many studies suggest a conformational change model of Chk1 activation in which phosphorylation shifts Chk1 from a closed inactive conformation to an open active conformation during the DNA damage response. However, no structural study has been reported to support this Chk1 activation model. Here we used FRET and bimolecular fluorescence complementary techniques to show that Chk1 indeed maintains a closed conformation in the absence of DNA damage through an intramolecular interaction between a region (residues 31-87) at the N-terminal kinase domain and the distal C terminus. A highly conserved Leu-449 at the C terminus is important for this intramolecular interaction. We further showed that abolishing the intramolecular interaction by a Leu-449 to Arg mutation or inducing ATR-dependent Chk1 phosphorylation by DNA damage disrupts the closed conformation, leading to an open and activated conformation of Chk1. These data provide significant insight into the mechanisms of Chk1 activation during the DNA damage response.
Insights
DNA damage response involves Chk1 activation. This study reveals Chk1 remains closed via intramolecular interactions until DNA damage or mutation opens it, activating the DNA damage checkpoint.
Area of Science:
- Molecular Biology
- Cellular Biology
- Biochemistry
Background:
- Phosphorylation of Chk1 by ATR is crucial for DNA damage checkpoint activation.
- The precise mechanism of Chk1 activation following phosphorylation remains incompletely understood.
- A conformational change model suggests Chk1 shifts from a closed to an open state upon activation.
Purpose of the Study:
- To investigate the structural basis of Chk1 activation.
- To provide experimental evidence for the conformational change model of Chk1 activation.
- To elucidate the role of intramolecular interactions in Chk1 regulation.
Main Methods:
- Förster Resonance Energy Transfer (FRET) techniques.
- Bimolecular fluorescence complementation (BiFC) assays.
- Site-directed mutagenesis (e.g., L449R mutation).
Main Results:
- Chk1 exists in a closed conformation in undamaged cells due to an intramolecular interaction between the N-terminal kinase domain (residues 31-87) and the C terminus.
- A conserved residue, Leucine-449 (Leu-449), is critical for maintaining this intramolecular interaction.
- DNA damage-induced ATR-dependent phosphorylation or disruption of the intramolecular interaction (L449R mutation) leads to a transition to an open, active Chk1 conformation.
Conclusions:
- The study provides structural evidence supporting the conformational change model of Chk1 activation.
- Intramolecular interactions regulate Chk1 conformation and activity.
- Understanding Chk1 conformational dynamics offers insights into DNA damage response pathways.
Related Concept Videos
02:36DNA Damage Can Stall the Cell Cycle
02:36DNA Damage can Stall the Cell Cycle
02:34Inhibition of Cdk Activity
02:37Restarting Stalled Replication Forks
02:19The Spindle Assembly Checkpoint
Many proteins function together to control the spindle assembly checkpoint. Mutations affecting these proteins may allow cells to proceed into anaphase prematurely, resulting in the...
02:15M-Cdk Drives Transition Into Mitosis
Cyclin-dependent kinases, or Cdks, work in concert with cyclins to control cell cycle transitions. M-Cdk, a complex of Cdk1 bound to M cyclin, is a well-known example of this coordinated control that drives the transition from the G2 to the M phase.
M cyclin...