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Updated: Apr 22, 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
Repair of a DNA-protein crosslink by replication-coupled proteolysis
Julien P Duxin1, James M Dewar1, Hasan Yardimci2
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Abstract:
DNA-protein crosslinks (DPCs) are caused by environmental, endogenous, and chemotherapeutic agents and pose a severe threat to genome stability. We use Xenopus egg extracts to recapitulate DPC repair in vitro and show that this process is coupled to DNA replication. A DPC on the leading strand template arrests the replisome by stalling the CMG helicase. The DPC is then degraded on DNA, yielding a peptide-DNA adduct that is bypassed by CMG. The leading strand subsequently resumes synthesis, stalls again at the adduct, and then progresses past the adduct using DNA polymerase ζ. A DPC on the lagging strand template only transiently stalls the replisome, but it too is degraded, allowing Okazaki fragment bypass. Our experiments describe a versatile, proteolysis-based mechanism of S phase DPC repair that avoids replication fork collapse.
Insights
DNA-protein crosslinks (DPCs) threaten genome stability but are repaired during DNA replication. This study reveals a proteolysis-dependent repair mechanism that allows replication forks to bypass DPCs, preventing fork collapse.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- DNA-protein crosslinks (DPCs) are DNA adducts formed by environmental, endogenous, or chemotherapeutic agents.
- DPCs pose a significant threat to genome stability by obstructing DNA replication and transcription.
- Efficient repair mechanisms are crucial to counteract the detrimental effects of DPCs.
Purpose of the Study:
- To investigate the in vitro mechanism of DNA-protein crosslink (DPC) repair using Xenopus egg extracts.
- To elucidate how DPC repair is coordinated with DNA replication.
- To understand how stalled replication forks handle DPCs on leading and lagging strands.
Main Methods:
- Utilized Xenopus egg extracts to create an in vitro system for studying DPC repair.
- Reconstituted DNA replication in the presence of DPCs to observe replisome dynamics.
- Employed biochemical assays to analyze DPC degradation and bypass by replication machinery.
Main Results:
- DPCs on the leading strand template stall the CMG helicase, leading to replisome arrest.
- DPCs are degraded on DNA, forming peptide-DNA adducts that are bypassed by CMG.
- DNA polymerase ζ facilitates replication past the peptide-DNA adducts on the leading strand.
- DPCs on the lagging strand template cause transient stalls, followed by degradation and Okazaki fragment bypass.
Conclusions:
- A versatile, proteolysis-dependent mechanism repairs DPCs during S phase.
- This repair pathway effectively prevents replication fork collapse in the presence of DPCs.
- The findings provide new insights into maintaining genome stability against DNA-protein crosslinking agents.
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