Replication bypass and mutagenic effect of α-deoxyadenosine site-specifically incorporated into single-stranded vectors

Abstract

α-2'-Deoxyadenosine (α) is a major adenine lesion produced by γ-ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing α were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for α were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site. The transfection assay revealed that α constituted a moderate block to DNA replication. The in vivo replication capacity to pass through α was ~ 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block. Similar data were obtained by in vitro replication of oligonucleotide templates containing α or F by E. coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing α was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of α introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated α was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G. A possible mechanism of the single nucleotide deletion associated with α is discussed on the basis of the misinsertion-strand slippage model.