Replication of an Oxidized Abasic Site in Escherichia coli by a dNTP-Stabilized Misalignment Mechanism that Reads Upstream and Downstream Nucleotides

Abstract

Abasic sites (AP) and oxidized abasic lesions are often referred to as noninstructive lesions because they cannot participate in Watson-Crick base pairing. The aptness of the term noninstructive for describing AP site replication has been called into question by recent investigations in E. coli using single-stranded shuttle vectors. These studies revealed that the replication of templates containing AP sites or the oxidized abasic lesions resulting from C1′-(L) and C4′-oxidation (C4-AP) are distinct from one another, suggesting that structural features other than Watson-Crick hydrogen bonds contribute to controlling replication. The first description of the replication of the abasic site resulting from formal C2′-oxidation (C2-AP) is presented here. Full-length and single-nucleotide deletion products are observed when templates containing C2-AP are replicated in E. coli. Single nucleotide deletion formation is largely dependent upon the concerted effort of pol II and pol IV, whereas pol V suppresses frameshift product formation. Pol V utilizes the A-rule when bypassing C2-AP. In contrast, pol II and pol IV utilize a dNTP-stabilized misalignment mechanism to read the upstream and downstream nucleotides when bypassing C2-AP. This is the first example in which the identity of the 3′-adjacent nucleotide is read during the replication of a DNA lesion. The results raise further questions as to whether abasic lesions are noninstructive lesions. We suggest that abasic site bypass is affected by the local biopolymer structure in addition to the structure of the lesion.Abasic sites (AP 1 ), the most common DNA lesions formed, are produced in a variety of ways in DNA, including as intermediates during base excision repair (1-3). In addition, a variety of oxidized abasic lesions are produced when DNA is exposed to oxidative stress. DNA damaging agents selectively produce oxidized abasic lesions, including 2-deoxyribonolactone (L), and the C4′-oxidized abasic site (C4-AP) (Figure 1) (4-7). These agents take advantage of the accessibility and/or relatively low bond strength of the respective carbon-hydrogen bonds (8-10). The C2′-carbon-hydrogen bond is the strongest of the deoxyribose bonds in DNA. Consequently, the C2-AP lesion is only produced by highly reactive species, presumably via † Supported by NIGMS grants GM-063028 to M.M.G. and ES-012259 and GM-021422 to M.F.G. * To whom correspondence should addressed. . E-mail: mgreenberg@jhu.edu.. ‡ Johns Hopkins University. § University of Southern California.SUPPORTING INFORMATION AVAILABLE ESI-MS of the oligonucleotides (3) containing C2-AP used to prepare the single-stranded plasmids and a figure describing the REAP experiment. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: C2′-oxidized abasic site, C2-AP; C4′-oxidized abasic site, C4-AP; abasic site, AP; 2-deoxyribonolactone, L; tetrahydrofuran abasic site model, F; polyacrylamide gel electrophoresis, PAGE; DNA polymerase II, pol II; DNA polymerase IV, pol IV; DNA...

The Substrate Specificity of MutY for Hyperoxidized Guanine Lesions in Vivo

“…This frameshift mutation was observed previously for the THF lesion in a 5′-TXG-3′ context, and even higher amounts of this -1 product were obtained when the lesion was 3′ to C (61). Furthermore, recent work from Kroeger and co-workers has shown that replication of a C2′ oxidized abasic site in E. coli relies on a dNTP-stablized primer/template misalignment mechanism to read the bases both upstream and downstream from the abasic site (62). This underscores the importance of the nucleotides surrounding a lesion in determining the outcome of translesion synthesis.…”

Excision of a Lyase-Resistant Oxidized Abasic Lesion from DNA

“…The C2′-oxidized abasic site induces mutations in DNA during replication in E. coli via an unusual mechanism that involves the upstream nucleotide (7). Hence, it is imperative that this lesion be repaired.…”

Translesion DNA Synthesis

“…This includes (but is not limited to) N 2 -acetylaminofluorene (AAF) and N 2 -aminofluorene (AF) modified guanines (80, 162, 177, 226), benzo[ a ]pyrene diol epoxide (BaP) guanine and adenine adducts (129, 162, 208), 8-oxoguanine (8-oxoG) and other guanine oxidation products (141, 163, 164), oxidized abasic lesions (118), N 2 -dG and other acrolein-induced and related adducts (102, 103, 157, 265), butadiene-induced guanine intrastrand and N 2 -N 2 -guanine interstrand crosslinks (41, 121), and alkylated bases (52). By using the single-lesion containing vectors in in vitro reconstituted reactions and by introducing them into strains lacking one or more TLS polymerase, it is straightforward to determine the contribution of each polymerase to the efficiency and accuracy of in vivo lesion bypass.…”

“…Evidence for the concerted action of SOS-inducible polymerases is also found when assessing TLS of an oxidized abasic site. When the lesion is replicated in E. coli , pol V incorporates dA opposite this lesion producing full-length replication products, while pol II and pol IV utilize a dNTP-stabilized misalignment mechanism to create single-nucleotide deletion products (118). …”