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Publication Detail

Title: Characterization of Interstrand DNA-DNA Cross-Links Derived from Abasic Sites Using Bacteriophage ϕ29 DNA Polymerase.

Authors: Yang, Zhiyu; Price, Nathan E; Johnson, Kevin M; Gates, Kent S

Published In Biochemistry, (2015 Jul 14)

Abstract: Interstrand cross-links in cellular DNA are highly deleterious lesions that block transcription and replication. We recently characterized two new structural types of interstrand cross-links derived from the reaction of abasic (Ap) sites with either guanine or adenine residues in duplex DNA. Interestingly, these Ap-derived cross-links are forged by chemically reversible processes, in which the two strands of the duplex are joined by hemiaminal, imine, or aminoglycoside linkages. Therefore, understanding the stability of Ap-derived cross-links may be critical in defining the potential biological consequences of these lesions. Here we employed bacteriophage φ29 DNA polymerase, which can couple DNA synthesis and strand displacement, as a model system to examine whether dA-Ap cross-links can withstand DNA-processing enzymes. We first demonstrated that a chemically stable interstrand cross-link generated by hydride reduction of the dG-Ap cross-link completely blocked primer extension by φ29 DNA polymerase at the last unmodified nucleobase preceding cross-link. We then showed that the nominally reversible dA-Ap cross-link behaved, for all practical purposes, like an irreversible, covalent DNA-DNA cross-link. The dA-Ap cross-link completely blocked progress of the φ29 DNA polymerase at the last unmodified base before the cross-link. This suggests that Ap-derived cross-links have the power to block various DNA-processing enzymes in the cell. In addition, our results reveal φ29 DNA polymerase as a tool for detecting the presence and mapping the location of interstrand cross-links (and possibly other lesions) embedded within regions of duplex DNA.

PubMed ID: 26103998 Exiting the NIEHS site

MeSH Terms: Bacillus Phages/enzymology*; Bacillus Phages/metabolism; Base Sequence; DNA-Directed DNA Polymerase/metabolism*; DNA/chemistry*; DNA/metabolism*; Molecular Sequence Data; Nucleic Acid Conformation; Oxidation-Reduction; Substrate Specificity

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