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Updated: Jan 28, 2026
A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells
Published on: March 5, 2018
TRAIP is a master regulator of DNA interstrand crosslink repair
R Alex Wu1, Daniel R Semlow1, Ashley N Kamimae-Lanning2
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
Abstract:
Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.
Insights
TRAIP controls DNA repair pathway choice for interstrand crosslinks. It recruits NEIL3 glycosylase or triggers Fanconi anaemia pathway repair via CMG helicase ubiquitylation, ensuring genome stability.
Area of Science:
- Molecular Biology
- DNA Repair Mechanisms
- Cellular Response to DNA Damage
Background:
- DNA interstrand crosslinks (ICLs) pose significant threats to genome integrity by blocking replication and transcription.
- Cells employ distinct pathways to repair ICLs, with pathway choice impacting repair fidelity and potential for genomic instability.
- The prioritization of ICL repair pathways, particularly the NEIL3 glycosylase versus Fanconi anaemia pathways, remained unclear.
Purpose of the Study:
- To elucidate the regulatory mechanism governing the choice between different DNA interstrand crosslink repair pathways.
- To identify the role of the E3 ubiquitin ligase TRAIP in replication-coupled ICL repair.
- To understand how TRAIP influences the recruitment of repair factors and the resolution of stalled replication forks.
Main Methods:
- Utilized Xenopus egg extracts to study replication fork collision with ICLs.
- Investigated the ubiquitylation activity of TRAIP on the CMG replicative helicase.
- Analyzed the differential recruitment of NEIL3 and the requirement for p97 ATPase in distinct repair outcomes.
Main Results:
- TRAIP is essential for both the NEIL3 and Fanconi anaemia repair pathways.
- TRAIP ubiquitylates the CMG helicase, with short ubiquitin chains recruiting NEIL3 and longer chains facilitating CMG unloading by p97.
- This differential ubiquitylation by TRAIP dictates the cell's choice between direct cleavage or homologous recombination repair of ICLs.
Conclusions:
- TRAIP acts as a master regulator, controlling the switch between ICL repair pathways.
- TRAIP's regulation of CMG ubiquitylation and unloading is critical for resolving replication-associated DNA damage.
- These findings provide crucial insights into maintaining genome stability during DNA replication stress.
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