title: Characterization of Interstrand DNA-DNA Cross-Links Derived from Abasic Sites Using Bacteriophage ϕ29 DNA Polymerase.
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.
authors.affiliation: ‡Department of Chemistry and §Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States.
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: ‡Department of Chemistry and §Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States.
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Price
authors.affiliation: ‡Department of Chemistry and §Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Johnson
authors.affiliation: ‡Department of Chemistry and §Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States.
authors.forename: Kent S
authors.initials: KS
authors.lastname: Gates
mesh_terms: D017103:Bacillus Phages / Q000201:enzymology* / Q000378:metabolism; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D004247:DNA; D004259:DNA-Directed DNA Polymerase