Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication

Jung Eun Yeo, Susith S Wickramaratne, Santoshkumar Khatwani, Yen Chih Wang, Jeffrey Vervacke, Mark D Distefano, Natalia Y Tretyakova

Research output: Contribution to journalArticle

26 Citations (Scopus)

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 l. 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.

Original languageEnglish (US)
Pages (from-to)1860-1868
Number of pages9
JournalACS Chemical Biology
Volume9
Issue number8
DOIs
StatePublished - Aug 15 2014

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DNA Replication
DNA
Proteins
Peptides
Alkynes
Uracil
Azides
Cycloaddition Reaction
DNA-Directed DNA Polymerase
Mutagenesis
Ionizing Radiation
Green Fluorescent Proteins
link protein
Cycloaddition
DNA Repair
Antineoplastic Agents
Ionizing radiation
Biomolecules
Free Radicals
Substrates

Cite this

Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication. / Yeo, Jung Eun; Wickramaratne, Susith S; Khatwani, Santoshkumar; Wang, Yen Chih; Vervacke, Jeffrey; Distefano, Mark D; Tretyakova, Natalia Y.

In: ACS Chemical Biology, Vol. 9, No. 8, 15.08.2014, p. 1860-1868.

Research output: Contribution to journalArticle

Yeo, Jung Eun ; Wickramaratne, Susith S ; Khatwani, Santoshkumar ; Wang, Yen Chih ; Vervacke, Jeffrey ; Distefano, Mark D ; Tretyakova, Natalia Y. / Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication. In: ACS Chemical Biology. 2014 ; Vol. 9, No. 8. pp. 1860-1868.
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