DNA Polymerase V Allows Bypass of Toxic Guanine Oxidation Products in Vivo

Abstract

Reactive oxygen and nitrogen radicals produced during metabolic processes, such as respiration and inflammation, combine with DNA to form many lesions primarily at guanine sites. Understanding the roles of the polymerases responsible for the processing of these products to mutations could illuminate molecular mechanisms that correlate oxidative stress with cancer. Using M13 viral genomes engineered to contain single DNA lesions and Escherichia coli strains with specific polymerase (pol) knockouts, we show that pol V is required for efficient bypass of structurally diverse, highly mutagenic guanine oxidation products in vivo. We also find that pol IV participates in the bypass of two spiroiminodihydantoin lesions. Furthermore, we report that one lesion, 5-guanidino-4-nitroimidazole, is a substrate for multiple SOS polymerases, whereby pol II is necessary for error-free replication and pol V for error-prone replication past this lesion. The results spotlight a major role for pol V and minor roles for pol II and pol IV in the mechanism of guanine oxidation mutagenesis.The genome is continually damaged by spontaneously generated and environmental chemical agents. Many of the lesions formed in DNA strongly inhibit replicative polymerases. If repair of these lesions fails or is not fast enough, cells can biochemically adapt to "tolerate" the toxic DNA lesions, as evidenced by replication of the damaged DNA (1). Human cells possess at least 15 DNA polymerases, including 5 that are referred to as translesion synthesis (TLS) 3 polymerases and are specifically involved in the replication of damaged DNA. Four of these enzymes, pol , pol , pol , and REV1, belong to the Y family of DNA polymerases, whereas pol belongs to the B family (2). Escherichia coli possess three polymerases, pol II, pol IV, and pol V, whose expression levels are up-regulated in response to DNA damage. Extensive research on the structure and function of these polymerases and their homologues suggests that these polymerases allow replication to progress in the presence of DNA lesions that are strongly inhibitory to the replicative polymerase, pol III (1). The presence of the three SOSinducible polymerases allows E. coli to be used as a model system for DNA replication past damage products.pol II (pol B) (3) is a B family DNA polymerase and has 3Ј 3 5Ј-exonuclease activity. Consequently, this enzyme replicates normal DNA with high fidelity and has an error rate less of than 10 Ϫ6 (4). pol II participates in error-free replication restart (5, 6) and can also carry out TLS past abasic sites (3, 7), 3,N 4 -ethenocytosine (8), and N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (9). pol IV (DinB) (10) and pol V (UmuD 2 ЈC) (11) are members of the Y family of DNA polymerases, which have less sterically restrictive active sites than normal replicative polymerases (12) and lack exonuclease activity (1). The error rates of these polymerases are much higher than the other E. coli polymerases and are on the order of 10 Ϫ4 to 10 Ϫ5 for pol IV (13) and 10 Ϫ3 to 10 Ϫ4...

Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions

“…The mixtures were transformed into SOS-induced, electrocompetent wild-type AB1157 E. coli cells and the isogenic E. coli cells that are deficient in Pol II, Pol IV, Pol V or all three polymerases, following the previously published procedures (23). In this vein, the SOS system was induced by irradiating the E. coli cells with 254 nm light at a dose of 45 J/m 2 , as described previously (24). We chose to conduct the replication experiments in SOS-induced cells because of the relatively low bypass efficiencies for most of the O 2 -alkyldT lesions ( vide infra ), and we found that the bypass efficiency for O 2 -EtdT in wild-type AB1157 cells was elevated by ∼3-fold upon SOS induction.…”

The Aminoglycoside Antibiotic Kanamycin Damages DNA Bases in Escherichia coli: Caffeine Potentiates the DNA-Damaging Effects of Kanamycin while Suppressing Cell Killing by Ciprofloxacin in Escherichia coli and Bacillus anthracis

“…In addition to the glycosylases, cells lacking one of three other repair proteins (AlkB, PhrB, SmbC [see below]) display greatly increased sensitivity. When the number of DNA replication-blocking lesions exceeds the capacity of the repair proteins involved in the direct removal of lesions, a third level of protection from killing ensues: translesion synthesis mediated by Pol V (56, 64) occurs, as shown here by the increased sensitivity to kanamycin of umuC mutants deficient in Pol V. Note that Pol V has been shown to allow bypass of toxic guanine oxidation products (50). (The role of dinB-encoded Pol IV translesion synthesis [29,66] here is less clear [ Fig.…”

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.…”