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Updated: Apr 15, 2026
Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
Published on: November 5, 2012
REV1 is important for the ATR-Chk1 DNA damage response pathway in Xenopus egg extracts
Darla DeStephanis1, Melissa McLeod1, Shan Yan1
1Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC 28223, USA.
Abstract:
The translesion DNA synthesis (TLS) polymerase REV1 is implicated in the bypass of the irreparable DNA damage such as interstrand crosslinks (ICLs). However, the potential role of REV1 in DNA damage response (DDR) pathway has not been determined. In this research communication, we provide evidence to demonstrate that REV1 plays a previously unidentified but important role in the ATR-Chk1 checkpoint activation in response to mitomycin C (MMC)-induced ICLs in Xenopus egg extracts. We further pinpointed that REV1 plays a downstream role of a checkpoint protein complex assembly including ATR, ATRIP, TopBP1 and the Rad9-Rad1-Hus1 complex to MMC-induced ICLs on chromatin in the DDR pathway. Notably, domain dissection analysis demonstrates that a C-terminal domain, but not the individual ubiquitin binding motifs, of REV1 is important for the binding of REV1 to MMC-damaged chromatin and the MMC-induced Chk1 phosphorylation. Yet, the ATR-Chk1 DDR pathway appears to be dispensable for the preferential association of REV1 to MMC-damaged chromatin. Taken together, REV1 is important for the DDR pathway in Xenopus egg extracts.
Insights
The DNA repair polymerase REV1 is crucial for activating the ATR-Chk1 DNA damage response pathway after interstrand crosslinks. REV1
Area of Science:
- DNA repair
- Cellular response to DNA damage
- Biochemistry
Background:
- Translesion DNA synthesis (TLS) polymerase REV1 is known to bypass irreparable DNA damage like interstrand crosslinks (ICLs).
- The specific role of REV1 within the broader DNA damage response (DDR) pathway remained largely undetermined.
Purpose of the Study:
- To investigate the previously unidentified role of REV1 in the DDR pathway.
- To determine REV1's involvement in checkpoint activation following ICLs.
Main Methods:
- Utilized Xenopus egg extracts to study the DDR pathway.
- Employed mitomycin C (MMC) to induce ICLs.
- Performed domain dissection analysis of REV1.
- Assessed checkpoint protein complex assembly and chromatin binding.
Main Results:
- REV1 plays a significant role in ATR-Chk1 checkpoint activation in response to MMC-induced ICLs.
- REV1 functions downstream of checkpoint protein complex assembly (ATR, ATRIP, TopBP1, Rad9-Rad1-Hus1) on chromatin.
- A C-terminal domain of REV1, not its ubiquitin binding motifs, is essential for chromatin binding and Chk1 phosphorylation.
- The ATR-Chk1 pathway is not required for REV1's preferential association with damaged chromatin.
Conclusions:
- REV1 is a critical component of the DNA damage response pathway in Xenopus egg extracts.
- REV1's C-terminal domain mediates its interaction with damaged chromatin, facilitating checkpoint activation.
- REV1's function in DDR is independent of the ATR-Chk1 pathway for initial chromatin localization.
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