Abstract
Adozelesin is a member of a family of extraordinarily cytotoxic DNA damaging agents that bind to the DNA minor groove in a sequence-specific manner and form covalent adducts with adenines. Previous studies employing purified enzymes and adozelesin-modified template DNAs suggested that adozelesin-DNA adducts inhibit DNA replication at the level of nascent DNA chain elongation. In this study, neutral/neutral two-dimensional agarose gel electrophoresis was employed to analyze simian virus 40 (SV40) DNA replication intermediates recovered from adozelesin-treated SV40 virus-infected cells. SV40 replication intermediates rapidly disappeared from infected cells when they were treated with adozelesin, but not when the cells were also treated with aphidicolin to block maturation of replicating SV40 DNA. We conclude that the disappearance of SV40 replication intermediates induced by adozelesin treatment was a consequence of maturation of these intermediates in the absence of new initiation events. Adozelesin inhibition of nascent chain elongation is first observed at concentrations above those needed to block initiation. Adozelesin treatment inhibits SV40 DNA replication at concentrations that produce adducts on just a small fraction of the intracellular population of SV40 DNA molecules.
Highlights
Minor groove in a sequence-specific manner and form covalent adducts with adenines
Adozelesin Effects on Initiation of simian virus 40 (SV40) DNA Replication—To directly examine the effect of adozelesin treatment on SV40 DNA replication, SV40-infected BSC-1 cells were treated with various concentrations of adozelesin for 2 h beginning 24 h after infection
To determine if the inhibitory effect of adozelesin on intracellular SV40 DNA replication occurs at the level of initiation or elongation of nascent DNA chains, we employed a neutral/ neutral two-dimensional agarose gel electrophoresis technique for analyzing DNA replication intermediates (27)
Summary
Minor groove in a sequence-specific manner and form covalent adducts with adenines. Previous studies employing purified enzymes and adozelesin-modified template DNAs suggested that adozelesin-DNA adducts inhibit DNA replication at the level of nascent DNA chain elongation. The indole and benzofuran substituents of adozelesin (Fig. 1, subunits B and C, respectively) form non-covalent interactions with DNA that may contribute to the sequence preference of the drug (11, 17–22). One possible explanation for this inhibitory effect is that DNA adducts formed by adozelesin and other alkylating agents directly inhibit replication fork progression at the site of adduct formation. This model is supported by cell-free studies, which show that the Adozelesin treatment inhibits SV40 DNA replication at presence of adozelesin-DNA adducts blocks the progression of concentrations that produce adducts on just a small fraction of the intracellular population of SV40 DNA molecules. The examination of DNA damage and drug effects on DNA replication is facilitated by the small (5 kilobase pairs) circular structure of SV40 and the high copy number to which it replicates in infected cells
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