Effects of proflavin and photoactivated proflavin on the template function of single-stranded DNA

DNA context-specific effects of the association of proflavin, single-stranded DNA and DNA polymerase on DNA polymerization reactions were examined. Frameshift mutations induced by the presence of proflavin during in vitro DNA replication of a single-stranded DNA template by the Klenow fragment of Escherichia coli DNA polymerase I were sequenced. More than 80% of the frameshifts were one base-pair deletions opposite purine bases that were immediately 3' to pyrimidines. Purines (Pu) that were not adjacent to pyrimidines (Py) were not deletion sites. The remaining deletions were opposite template pyrimidines that were also immediately 3' to another pyrimidine. All pyrimidine site deletions occurred in the context 5' PyPyPu 3'. In additional experiments, the site-specific inhibition of processive DNA polymerization by proflavin was examined. A novel inhibition of polymerization was found opposite all pyrimidines in the template when proflavin-template complexes were exposed to ten seconds of white light. This inhibition of polymerization is reversible. Longer photoactivation led to an altered pattern of DNA sequence-specific inhibition that was not reversible. The role of DNA sequence-specific interactions of proflavin with DNA in proflavin mutagenesis is discussed.