Error-Prone Translesion DNA Synthesis by Escherichia coli DNA Polymerase IV (DinB) on Templates Containing 1,2-dihydro-2-oxoadenine

Abstract

Escherichia coli DNA polymerase IV (Pol IV) is involved in bypass replication of damaged bases in DNA. Reactive oxygen species (ROS) are generated continuously during normal metabolism and as a result of exogenous stress such as ionizing radiation. ROS induce various kinds of base damage in DNA. It is important to examine whether Pol IV is able to bypass oxidatively damaged bases. In this study, recombinant Pol IV was incubated with oligonucleotides containing thymine glycol (dTg), 5-formyluracil (5-fodU), 5-hydroxymethyluracil (5-hmdU), 7,8-dihydro-8-oxoguanine (8-oxodG) and 1,2-dihydro-2-oxoadenine (2-oxodA). Primer extension assays revealed that Pol IV preferred to insert dATP opposite 5-fodU and 5-hmdU, while it inefficiently inserted nucleotides opposite dTg. Pol IV inserted dCTP and dATP opposite 8-oxodG, while the ability was low. It inserted dCTP more effectively than dTTP opposite 2-oxodA. Pol IV's ability to bypass these lesions decreased in the order: 2-oxodA > 5-fodU~5-hmdU > 8-oxodG > dTg. The fact that Pol IV preferred to insert dCTP opposite 2-oxodA suggests the mutagenic potential of 2-oxodA leading to A:T→G:C transitions. Hydrogen peroxide caused an ~2-fold increase in A:T→G:C mutations in E. coli, while the increase was significantly greater in E. coli overexpressing Pol IV. These results indicate that Pol IV may be involved in ROS-enhanced A:T→G:C mutations.

Figure 1

The structures of the studied oxidatively damaged bases, thymine glycol (dTg), 5-formyluracil (5-fodU), 5-hydroxymethyluracil (5-hmdU), 1,2-dihydro-2-oxoadenine (2-oxodA), and 7,8-dihydro-8-oxoguanine (8-oxodG).

Figure 2

Primer extension assay for Pol IV to bypass dTg, 5-fodU, and 5-hmdU in the template oligonucleotides. Primers were labeled at the 5′-terminal by polynucleotide kinase and annealed with appropriate template oligonucleotide. Primer/templates (50 fmol) in a 10-μl reaction mixture was incubated at 20°C with purified Pol IV at 50 nM for 30 min (left) and at 200 nM for 12 hr (right), followed by polyacrylamide gel electrophoresis. (a) dT (primer 1/template 8), (b) dTg (primer 1/template 9), (c) 5-fodU (primer 2/template 6), and (d) 5-hmdU (primer 2/template 7).

Figure 3

Primer extension assay for Pol IV to bypass 8-oxodG and 2-oxodA in the template oligonucleotide. Primer 3 was labeled at the 5′-terminal by polynucleotide kinase and annealed with appropriate template oligonucleotides. Primer 3/templates (50 fmol) in a 10-μl reaction mixture were incubated at 20°C with purified Pol IV at 50 nM for 30 min (left) and at 200 nM for 12 hr (right), followed by polyacrylamide gel electrophoresis. (a) 8-oxodG (primer 3/template 4), (b) 2-oxodA (primer 3/template 2).

Figure 4

Possible wobble structure for base pairing of the 1,2-dihydro-2-oxoadenine (2-oxodA):dC.