Termination sites of the in vitro nick-translation reaction on DNA that had photoreacted with psoralen

Abstract

A double-stranded circular DNA having a single nick at a specific site has been photochemically induced to react with 4'-hydroxymethyl-4,5', 8-trimethylpsoralen (HMT) and used as a substrate for nick-translation with Escherichia coli DNA polymerase I holoenzyme. By using the dideoxy chain-terminating sequencing procedure, it was possible to map the termination sites observed on the template that had photoreacted with HMT. These sites occur at nucleotides preceding potential psoralen crosslinking sites. Analysis of DNA products synthesized on templates that had photoreacted under conditions designed to maximize psoralen monoaddition revealed that DNA polymerase I is not stopped by this lesion. Psoralen monoadducts situated on the template strand act only as kinetic attenuators, whereas psoralen monoadducts localized on the nick-translated strand have no effect on the rate of synthesis. These data suggest that psoralen crosslinks are responsible for the lethal effects of psoralen photochemistry in E. coli. Mutagenesis may be associated, however, with the repair replication of psoralen monoadducts by E. coli DNA polymerase I.