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Updated: Apr 28, 2026
Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System
Published on: August 21, 2016
Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication
Jung Eun Yeo1, Susith Wickramaratne, Santoshkumar Khatwani
1Masonic Cancer Center and Departments of †Medicinal Chemistry and ‡Chemistry, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Abstract:
DNA-protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA-protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts.
Insights
DNA-protein cross-links (DPCs) block DNA replication. Larger lesions halt DNA polymerases, suggesting proteolytic degradation may be needed for repair. This research advances understanding of DNA damage responses.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA-protein cross-links (DPCs) are significant DNA lesions caused by toxins, radiation, and drugs.
- DPCs impede essential DNA processes like replication, transcription, and repair, potentially causing mutations and cell death.
- Understanding DPCs is limited by challenges in creating specific, defined DPC models.
Purpose of the Study:
- To develop a method for creating site-specific DNA-protein cross-links (DPCs).
- To investigate how human DNA polymerases bypass or stall at these DPC lesions.
- To understand the biological consequences of DPCs on DNA replication.
Main Methods:
- Generated site-specific DPCs using copper-catalyzed click chemistry between alkyne-modified DNA and azide-modified proteins/peptides.
- Assessed in vitro primer extension using human lesion bypass DNA polymerases (η, κ, ν, ι).
- Utilized green fluorescent protein and 10-mer/23-mer peptides for cross-linking.
Main Results:
- DNA replication was completely blocked by DPCs involving green fluorescent protein and a 23-mer peptide.
- DNA replication was successfully bypassed at a DPC site involving a 10-mer peptide.
- Human lesion bypass DNA polymerases showed differential responses to DPC size and protein moiety.
Conclusions:
- The size and nature of the protein moiety in DPCs significantly influence DNA polymerase bypass efficiency.
- Larger DPCs act as formidable blocks to DNA replication, exceeding the bypass capacity of tested polymerases.
- Proteolytic processing of DPCs may be a necessary step to facilitate replication restart and genome stability.
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