Effects of oxidized abasic DNA lesions on DNA repair and replication

In the cell, the interaction of reactive oxygen species or certain antitumor drugs with 2-deoxyribose produces a variety of oxidized abasic sites. Until recently little was known about the effects of oxidized abasic sites. However, the advent of synthetic methods that place a single abasic lesion site-specifically into an oligonucleotide has facilitated the study of these lesions. We have used these synthetic methods to examine the biological effects of three oxidized abasic DNA lesions: 2-deoxyribonolactone, the C4'-oxidized abasic site, and the C2'-oxidized abasic site. Studies were carried out both in vitro and in vivo using Escherichia coli .; In vitro analyses were directed at elucidating the interactions of each lesion with base excision repair enzymes. In vivo studies were carried out using shuttle vector technology and an Escherichia coli model system to investigate DNA replication past a single abasic lesion present in a single-stranded plasmid. Our studies were conducted using wild type Escherichia coli, as well as a variety of repair deficient and SOS-polymerase deficient cell lines (with and without SOS-induction). In addition, 2-deoxyribose and the model abasic lesion, tetrahydrofuran, were examined side-by-side for comparison with the oxidized abasic sites.; Use of the Restriction Endonuclease And Postlabeling assay allowed us to determine nucleotide incorporation preferences opposite each lesion, deletions produced upon bypass, and specific polymerase effects. We found that, while abasic sites are generally referred to as "non-instructional" lesions, each oxidized abasic site produces a distinctive pattern of mutations and is, therefore, biologically unique.