Error‐Prone Translesion DNA Synthesis byEscherichia 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.

The DinB Superfamily Includes Novel Mycothiol, Bacillithiol, and Glutathione S-Transferases

“…As pointed out previously (52) the DinB superfamily proteins are not to be confused with the E. coli dinB encoded type IV DNA polymerase and homologous enzymes that are involved in error prone DNA repair (53, 54). The first DinB superfamily protein to have a function assigned was the mycothiol-dependent maleylpyruvate isomerase from C. glutamicum (34, 35) and this defines the MDMPI_N family.…”

Epistatic Roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in Coping with Reactive Oxygen Species-Induced DNA Damage

“…Consistent with this model, E. coli DinB catalyzes incorporation of dCTP opposite template–2-hydroxyadenine, 2-hydroxyadenine opposite template-dG (or template-dT), and 8-oxo-dG opposite template-dA. If left unrepaired, these insertions will result in TA→CG, GC→TA or AT→CG substitutions, respectively, following the subsequent round of DNA replication [18], [51]. Nucleotide sequence analysis indicated that although the majority of H 2 O 2 -induced mutations corresponded to TA→CG transitions, this substitution represented less than one-half of the mutations in the Δ dinB strain (Fig.…”

Discrimination against the Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB

“…Indeed, a similar trend has been observed with DinB and 8-oxo-G, as DinB appears to be less efficient in replicating DNA templates containing 8-oxo-G than it is in incorporating the nucleotide 8-oxo-GTP. 48,49 Other polymerases can also exhibit a marked asymmetry between utilization of modified bases in the template and the incoming nucleotide or in the fidelity of base-pair formation. [50][51][52] This observation may reflect asymmetry in the polymerase active site, 52 suggesting that the local environment of the template base is apparently quite different from that of the incoming nucleotide, which could lead to differences in the efficiency of bond formation or translocation.…”