ABSTRACT
Cells exposed to DNA damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription. Luciferase gene expression analysis with DNA templates containing uracil or 8-oxoguanine, placed at a defined position, indicated that in nondividing Escherichia coli cells, efficient mutagenic lesion bypass does occur in vivo during transcription. Sequence analyses of the transcript population revealed that RNA polymerase inserts adenine opposite to uracil, and adenine or cytosine opposite to 8-oxoguanine. Surprisingly, deletions were also detected for 8-oxoguanine-containing templates, indicating RNA polymerase slippage over this lesion. Genetic analyses showed that, in E. coli, 8-oxoguanine is subject to transcription-coupled repair. Consequently, DNA damages alter transcription fidelity in vivo, which may lead to the production of mutant proteins that have the potential to change the phenotype of nondividing cells.
Subject(s)
DNA Damage/genetics , Escherichia coli/genetics , Guanine/analogs & derivatives , Guanine/pharmacology , Mutagenesis/genetics , Transcription, Genetic/genetics , Uracil/pharmacology , Base Sequence/drug effects , Base Sequence/genetics , DNA Repair/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/drug effects , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Bacterial/genetics , Mutagenesis/drug effects , Phenotype , Transcription, Genetic/drug effects , Uracil/metabolismABSTRACT
We have previously reported the synthesis of vinylphosphonate-linked thymidine dimers and their incorporation into synthetic oligonucleotides to create vinylphosphonate internucleotide linkages in the DNA. Such linkages have a profound effect on DNA backbone rotational flexibility, and we have shown that the PcrA helicase, which requires such flexibility, is inhibited when it encounters these linkages on the translocating strand. In this study, we have investigated the effects of these linkages on the dsDNA specific exonuclease III and on the ssDNA specific mung bean nuclease to establish whether our modification confers resistance to nucleases making it suitable for antisense therapy applications. We also investigated the effect on DNA polymerase I to establish whether we could in the future use this enzyme to incorporate these linkages in the DNA. Our results show that a single modification does not affect the activity of DNA polymerase I, but four vinylphosphonate linkages in tandem inhibit its activity. Furthermore, such linkages do not confer significant nuclease resistance to either exonuclease III or mung bean nuclease, but unexpectedly, they alter the cleavage specificity of exonuclease III.
