Characterization of Interstrand DNA–DNA Cross-Links
Derived from Abasic Sites Using Bacteriophage ϕ29 DNA Polymerase
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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