Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

When a high-fidelity DNA polymerase encounters certain DNA-damage sites, its progress can be stalled and one or more lesion-bypass polymerases are recruited to transit the lesion. Here, we consider two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine (8-oxoG), a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene, in the high-fidelity DNA polymerase Bacillus fragment (BF) from Bacillus stearothermophilus and in the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. The tight fit of the BF polymerase around the nascent base pair contrasts with the more spacious, solvent-exposed active site of Dpo4, and these differences in architecture result in distinctions in their respective functions: one-step versus stepwise polymerase translocation, mutagenic versus accurate bypass of 8-oxoG, and polymerase stalling versus mutagenic bypass at bulky benzo[a]pyrene-derived lesions.     

The N-clasp of human DNA polymerase kappa promotes blockage or error-free bypass of adenine- or guanine-benzo[a]pyrenyl lesions

DNA bypass polymerases are utilized to transit bulky DNA lesions during replication, but the process frequently causes mutations. The structural origins of mutagenic versus high fidelity replication in lesion bypass is therefore of fundamental interest. As model systems, we investigated the molecular basis of the experimentally observed essentially faithful bypass of the guanine 10S-(+)-trans-anti-benzo[a]pyrene-N²-dG adduct by the Y-family human DNA polymerase κ, and the observed blockage of pol κ produced by the adenine 10S-(+)-trans-anti-benzo[a]pyrene-N²-dA adduct. These lesions are derived from the most tumorigenic metabolite of the ubiquitous cancer-causing pollutant, benzo[a]pyrene. We compare our results for the dG adduct with our earlier studies for the pol κ archaeal homolog Dpo4, which processes the same lesion in an error-prone manner. Molecular modeling, molecular mechanics calculations and molecular dynamics simulations were utilized. Our results show that the pol κ N-clasp is a key structural feature that accounts for the dA adduct blockage and the near-error-free bypass of the dG lesion. Absence of the N-clasp in Dpo4 explains the error-prone processing of the same lesion by this enzyme. Thus, our studies elucidate structure-function relationships in the fidelity of lesion bypass.     

Accurate representation of B-DNA double helical structure with implicit solvent and counterions

High-resolution nuclear magnetic resonance (NMR) and crystallographic data have been taken to refine the force field used in the torsion angle space nucleic acids molecular mechanics program DUPLEX. The population balance deduced from NMR studies of two carcinogen-modified DNA conformers in equilibrium was used to fine tune a sigmoidal, distance-dependent dielectric function so that reasonable relative energies could be obtained. In addition, the base-pair and backbone geometry from high-resolution crystal structures of the Dickerson-Drew dodecamer was used to re-evaluate the deoxyribose pseudorotation profile and the Lennard-Jones nonbonded energy terms. With a modified dielectric function that assumes a very steep distance-dependent form, a deoxyribose pseudorotation profile with reduced energy barriers between C2'- and C3'-endo minima, and a shift of the Lennard-Jones potential energy minimum to a distance approximately 0.4 A greater than the sum of the van der Waals' radii, the sequence-dependent conformational features of the Dickerson-Drew dodecamer in both the solid state and the aqueous liquid crystalline phase are well reproduced. The robust performance of the revised force field, in conjunction with its efficiency through implicit treatment of solvent and counterions, provides a valuable tool for elucidating conformations and structure-function relationships of DNA, including those of molecules modified by carcinogens and other ligands.     

4-hydroxyequilenin-adenine lesions in DNA duplexes: stereochemistry, damage site, and structure

The equine estrogens, equilin and equilenin, are major components of the drug Premarin, the most widely used formula for hormone replacement therapy. The derivative 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react in vitro and in vivo with cytosine and adenine in DNA. Unique cyclic adducts containing the same bicyclo[3.3.1]nonane-type connection ring are produced. Each base adduct has four stereoisomers. In order to elucidate the structural effects of A versus C modification, we have carried out molecular dynamics simulations of the stereoisomeric 4-OHEN-A adducts in DNA 11-mer duplexes and compared results with an earlier study of the C adducts (Ding, S., Shapiro, R., Geacintov, N.E., and Broyde, S. (2005) Equilenin-Derived DNA Adducts to Cytosine in DNA Duplexes: Structures and Thermodynamics, Biochemistry 44, 14565-14576). Similar stereochemical principles govern the orientations in DNA duplexes of the 4-OHEN-A adducts as for the analogous C adducts, with opposite orientations of the equilenin rings in stereoisomeric pairs of adducts characterized by near-mirror image circular dichroism (CD) spectra. However, the larger purine adducts have unique structural properties in the duplexes that distinguish their characteristics from those of the pyrimidine adducts. Significant differences are observed in terms of hydrogen bonding, stacking, bending, groove dimensions, solvent exposure, and hydrophobic interactions; also, each of the four stereoisomeric 4-OHEN-A adducts exhibit distinct structural features. Each base adduct and stereoisomer distorts the structure of the DNA duplex differently. These characteristics may manifest themselves in terms of differential nucleotide excision repair susceptibilities and mutagenic activities of the 4-OHEN-A and C adducts.     

Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis

Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.     

Flexible 5-guanidino-4-nitroimidazole DNA lesions: structures and thermodynamics

5-Guanidino-4-nitroimidazole (NI), derived from guanine oxidation by reactive oxygen and nitrogen species, contains an unusual flexible ring-opened structure, with nitro and guanidino groups which possess multiple hydrogen bonding capabilities. In vitro primer extension experiments with bacterial and mammalian polymerases show that NI incorporates C as well as A and G opposite the lesion, depending on the polymerase. To elucidate structural and thermodynamic properties of the mutagenic NI lesion, we have investigated the structure of the modified base itself and the NI-containing nucleoside with high-level quantum mechanical calculations and have employed molecular modeling and molecular dynamics simulations in solution for the lesion in B-DNA duplexes, with four partner bases opposite the NI. Our results show that NI adopts a planar structure at the damaged base level. However, in the nucleoside and in DNA duplexes, steric hindrance between the guanidino group and its linked sugar causes NI to be nonplanar. The NI lesion can adopt both syn and anti conformations on the DNA duplex level, with the guanidino group positioned in the DNA major and minor grooves, respectively; the specific preference depends on the partner base. On the basis of hydrogen bonding and stacking interactions, groove dimensions, and bending, we find that the least distorted NI-modified duplex contains partner C, consistent with observed incorporation of C opposite NI. However, hydrogen bonding interactions between NI and partner G or A are also found, which would be compatible with the observed mismatches.     

Mutagenic nucleotide incorporation and hindered translocation by a food carcinogen C8-dG adduct in Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): modeling and dynamics studies

Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics (MD) simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP adduct enhances incorporation of these mismatches. Finally, bulky C8-dG adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N²-dG adducts, which can reside on the minor groove side, appear to cause less hindrance when in this position.     

A new anti conformation for N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) allows Watson-Crick pairing in the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)

Primer extension studies have shown that the Y-family DNA polymerase IV (Dpo4) from Sulfolobus solfataricus P2 can preferentially insert C opposite N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG) [F. Boudsocq, S. Iwai, F. Hanaoka and R. Woodgate (2001) Nucleic Acids Res., 29, 4607–4616]. Our goal is to elucidate on a structural level how AAF-dG can be harbored in the Dpo4 active site opposite an incoming dCTP, using molecular modeling and molecular dynamics simulations, since AAF-dG prefers the syn glycosidic torsion. Both anti and syn conformations of the templating AAF-dG in a Dpo4 ternary complex were investigated. All four dNTPs were studied. We found that an anti glycosidic torsion with C1′-exo deoxyribose conformation allows AAF-dG to be Watson–Crick hydrogen-bonded with dCTP with modest polymerase perturbation, but other nucleotides are more distorting. The AAF is situated in the Dpo4 major groove open pocket with fluorenyl rings 3′- and acetyl 5′-directed along the modified strand, irrespective of dNTP. With AAF-dG syn, the fluorenyl rings are in the small minor groove pocket and the active site region is highly distorted. The anti-AAF-dG conformation with C1′-exo sugar pucker can explain the preferential incorporation of dC by Dpo4. Possible relevance of our new major groove structure for AAF-dG to other polymerases, lesion repair and solution conformations are discussed.