1-Nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG AP ). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dG AP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dG AP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.Environmental pollutants have been shown to impact human health at the molecular level. One detrimental route is the modification of genomic DNA and nucleotides (1). If DNA lesions are not recognized and removed by the cellular DNA repair machinery, they will stall replicative DNA polymerases (2-8).To rescue DNA replication, cells employ lesion bypass DNA polymerases to traverse unrepaired lesions. Most of these enzymes belong to the Y-family of DNA polymerases. The Y-family enzymes possess relatively flexible and solvent-accessible active sites to accommodate bulky DNA lesions (9, 10). However, Y-family DNA polymerases catalyze DNA synthesis over undamaged DNA with low fidelity and poor processivity (6, 10 -12). The Y-family DNA polymerases have been identified in all three domains of life, e.g. four in humans (DNA polymerases , , , and Rev1), two in Escherichia coli (DNA polymerases IV and V), and one in Sulfolobus solfataricus (Dpo4). Because Dpo4 can be expressed in E. coli and purified with a high yield, it has been extensively studied in vitro as a prototype Y-family enzyme. Dpo4 catalyzes DNA synthesis on an undamaged DNA template with a fidelity of one error per 1,000 -10,000 nucleotide incorporations based on presteady-state kinetic analysis from 37 to 56°C (13-15). Dpo4 is capable of bypassing a myriad of DNA lesions including apurinic/apyrimidinic (abasic) sites (16 -19), 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (20, 21), 1,N 2 -etheno(⑀)guanosine (22), cis-syn thymine-thymine dimer (23-...

3-Nitrobenzanthrone (3-NBA), a potent mutagen and suspected human carcinogen, is a common environmental pollutant. The genotoxicity of 3-NBA has been associated with its ability to form DNA adducts, including N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). To investigate the molecular mechanism of C8-dG-ABA mutagenesis in human cells, we have replicated a plasmid containing a single C8-dG-ABA in human embryonic kidney 293T (HEK293T) cells, which yielded 14% mutant progeny. The major types of mutations induced by C8-dG-ABA were G → T > G → A > G → C. siRNA knockdown of the translesion synthesis (TLS) DNA polymerases (pols) in HEK293T cells indicated that pol η, pol κ, pol ι, pol ζ, and Rev1 each have a role in replication across this adduct. The extent of TLS was reduced with each pol knockdown, but the largest decrease (of ∼55% reduction) in the level of TLS occurred in cells with knockdown of pol ζ. Pol η and pol κ were considered the major contributors of the mutagenic TLS, because the mutation frequency (MF) decreased by 70%, when these pols were simultaneously knocked down. Rev1 also is important for mutagenesis, as reflected by the 60% reduction in MF upon Rev1 knockdown, but it probably plays a noncatalytic role by physically interacting with the other two Y-family pols. In contrast, pol ζ appeared to be involved in the error-free bypass of the lesion, because MF increased by 60% in pol ζ knockdown cells. These results provide important mechanistic insight into the bypass of the C8-dG-ABA adduct.

8,5′-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5′S)-8,5′-cyclo-2′-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II– strain, whereas it increased considerably in a pol IV– strain. Remarkably, no progeny was recovered from a pol V– strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ∼34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5′C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, is formed in DNA by the reaction of thymine with reactive oxygen species. The 5R Tg lesion was incorporated site-specifically into 5′-d(G1T2G3C4G5Tg6G7T8T9T10G11T12)-3′; Tg = 5R Tg. The Tg-modified oligodeoxynucleotide was annealed with either 5′-d(A13C14A15A16A17C18A19C20G21C22A23C24)-3′, forming the Tg6•A19 base pair, corresponding to the oxidative damage of thymine in DNA, or 5′-d(A13C14A15A16A17C18G19C20G21C22A23C24)-3′, forming the mismatched Tg6•G19 base pair, corresponding to the formation of Tg following oxidative damage and deamination of 5-methylcytosine in DNA. At 30 °C, the equilibrium ratio of cis-5R,6S:trans-5R,6R epimers was 7:3 for the duplex containing the Tg6•A19 base pair. In contrast, for the duplex containing the Tg6•G19 base pair, the cis-5R,6S:trans-5R,6R equilibrium favored the cis-5R,6S epimer; the level of the trans-5R,6R epimer remained below the level of detection by NMR. The data suggested that Tg disrupted hydrogen bonding interactions, either when placed opposite to A19 or G19. Thermodynamic measurements indicated a 13 °C reduction of Tm regardless of whether Tg was placed opposite dG or dA in the complementary strand. Although both pairings increased the free energy of melting by 3 kcal/mol, the melting of the Tg•G pair was more enthalpically favored than was the melting of the Tg•A pair. The observation that the position of the equilibrium between the cis-5R,6S and trans-5R,6R thymine glycol epimers in duplex DNA was affected by the identity of the complementary base extends upon observations that this equilibrium modulates the base excision repair of Tg [Ocampo-HafallaM. T.; AltamiranoA.; BasuA. K.; ChanM. K.; OcampoJ. E.; CummingsA.Jr.; BoorsteinR. J.; CunninghamR. P.; TeeborG. W.DNA Repair (Amst)2006, 5, 444−454].