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.

Human RNA polymerase II is partially blocked by DNA adducts derived from tumorigenic benzo[c]phenanthrene diol epoxides: relating biological consequences to conformational preferences.

Environmental polycyclic aromatic hydrocarbons (PAHs) are metabolically activated to diol epoxides that can react with DNA, resulting in covalent modifications to the bases. The (+)- and (-)-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene (anti-BPhDE) isomers are diol epoxide metabolites of the PAH benzo[c]phenanthrene (BPh). These enantiomers readily react with DNA at the N6 position of adenine, forming bulky (+)-1R- or (-)-1S-trans-anti-[BPh]-N6-dA adducts. Transcription-coupled nucleotide excision repair clears such bulky adducts from cellular DNA, presumably in response to RNA polymerase transcription complexes that stall at the bulky lesions. Little is known about the effects of [BPh]-N6-dA lesions on RNA polymerase II, hence, the behavior of human RNA polymerase II was examined at these adducts. A site-specific, stereochemically pure [BPh]-N6-dA adduct was positioned on the transcribed or non-transcribed strand of a DNA template with a suitable promoter for RNA polymerase II located upstream from the lesion. Transcription reactions were then carried out with HeLa nuclear extract. Each [BPh]-dA isomer strongly impeded human RNA polymerase II progression when it was located on the transcribed strand; however, a small but significant degree of lesion bypass occurred, and the extent of polymerase blockage and bypass was dependent on the stereochemistry of the adduct. Molecular modeling of the lesions supports the idea that each adduct can exist in two orientations within the polymerase active site, one that permits nucleotide incorporation and another that blocks the RNA polymerase nucleotide entry channel, thus preventing base incorporation and causing the polymerase to stall or arrest.

Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct.

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)-trans-anti-[BP]-N(2)-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N(2)-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)-trans-anti-[BP]-N(2)-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5'-CG*T-3' sequence context. Regardless of the base positioned opposite (+)-trans-anti-[BP]-N(2)-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)-trans-anti-[BP]-N(2)-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3' terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson-Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)-trans-anti-[BP]-N(2)-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer-template duplex region, leaving critical protein-DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5'-CG*T-3' sequence can be rationalized by the stability of interactions between the polymerase protein, primer-template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)-trans-anti-[BP]-N(2)-dG in the +1 position (T > G > A > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = G > C). In addition, extension past (+)-trans-anti-[BP]-N(2)-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase-DNA interactions than it is within the active site itself. The dN:(+)-trans-anti-[BP]-N(2)-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein-DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.

Construction and purification of site-specifically modified DNA templates for transcription assays.

Chemical and physical agents can alter the structure of DNA by modifying the bases and the phosphate-sugar backbone, consequently compromising both replication and transcription. During transcription elongation, RNA polymerase complexes can stall at a damaged site in DNA and mark the lesion for repair by a subset of proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Although our understanding of DNA lesion effects on transcription elongation and the associated effects of stalled transcription complexes on DNA repair is broadening, the attainment of critical data is somewhat impeded by labor-intensive, time- consuming processes that are required to prepare damaged DNA templates. Here, we describe an approach for building linear DNA templates that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II. The method is rapid, making use of biotin-avidin interactions and paramagnetic particles to purify the final product. Data are supplied demonstrating that these templates support transcription, and we emphasize the potential versatility of the protocol and compare it with other published methods.

Analysis of protein sequence/structure similarity relationships.

Current analyses of protein sequence/structure relationships have focused on expected similarity relationships for structurally similar proteins. To survey and explore the basis of these relationships, we present a general sequence/structure map that covers all combinations of similarity/dissimilarity relationships and provide novel energetic analyses of these relationships. To aid our analysis, we divide protein relationships into four categories: expected/unexpected similarity (S and S(?)) and expected/unexpected dissimilarity (D and D(?)) relationships. In the expected similarity region S, we show that trends in the sequence/structure relation can be derived based on the requirement of protein stability and the energetics of sequence and structural changes. Specifically, we derive a formula relating sequence and structural deviations to a parameter characterizing protein stiffness; the formula fits the data reasonably well. We suggest that the absence of data in region S(?) (high structural but low sequence similarity) is due to unfavorable energetics. In contrast to region S, region D(?) (high sequence but low structural similarity) is well-represented by proteins that can accommodate large structural changes. Our analyses indicate that there are several categories of similarity relationships and that protein energetics provide a basis for understanding these relationships.

Toward understanding the mutagenicity of an environmental carcinogen: structural insights into nucleotide incorporation preferences.

Bulky carcinogen-DNA adducts, including (+)-trans-anti-[BP]-N(2)-dG derived from the reaction of (+)-anti-benzo[a]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be misreplicated. Experimental results have shown that nucleotides are inserted opposite the (+)-trans-anti-[BP]-N(2)-dG adduct by bacteriophage T7 DNA polymerase with the order of preference A>T>or=G>C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)-trans-anti-[BP]-N(2)-dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)-trans-anti-[BP]-N(2)-dG adduct adopt the syn conformation in each case to avoid deranging essential protein-DNA interactions. While the dATP: (+)-trans-anti-[BP]-N(2)-dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)-trans-anti-[BP]-N(2)-dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)-trans-anti-[BP]-N(2)-dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein-DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)-trans-anti-[BP]-N(2)-dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)-trans-anti-[BP]-N(2)-dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)-trans-anti-[BP]-N(2)-dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)-trans-anti-[BP]-N(2)-dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this carcinogen-DNA adduct in a model system.

DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistry-dependent manner.

Many carcinogens exert their cancer-causing effects by reacting with DNA either directly or following metabolic activation, resulting in covalently linked combination molecules known as carcinogen-DNA adducts. The presence of such lesions in the genome increases the error frequency of the replication machinery, causing mutations that contribute to the initiation and progression of cancer. Cellular DNA repair pathways remove carcinogen adducts from DNA, thus averting the mutagenic potential of many DNA lesions by reducing their presence in the genome. Bulky DNA adducts, like those derived from a number of activated environmental carcinogens such as polycyclic aromatic hydrocarbons (PAHs), are primarily repaired by the nucleotide excision repair (NER) pathway. Transcription-coupled NER (TC-NER) preferentially removes lesions from the transcribed strand of actively expressed genes, and RNA polymerase II stalled at the lesion quite possibly initiates the pathway. Among the bulky DNA adducts that are subject to TC-NER are those resulting from the reaction of the metabolically activated PAH benzo[a]pyrene (BP) with DNA. The P450 mixed-function oxygenases convert BP into a number of reactive intermediates, including tumorigenic (+)- and non-tumorigenic (-)-anti-benzo[a]pyrene diol epoxide (BPDE) that react with DNA via trans epoxide opening to form (+)-trans-anti-[BP]-N(2)-dG ((+)-ta[BP]G) and (-)-trans-anti-[BP]-N(2)-dG ((-)-ta[BP]G), respectively. To test the effect of these lesions on RNA synthesis, in vitro transcription assays using human nuclear extracts were performed with DNA templates containing an RNAPII promoter and a stereochemically pure (+)- or (-)-ta[BP]G adduct on the transcribed or non-transcribed strand. Transcription past (+)- or (-)-ta[BP]G adducts was investigated in the same sequence context to examine stereochemical effects. The (+)-ta[BP]G adduct was investigated in two different local sequence contexts to determine if the surrounding bases influence the adduct's ability to block transcription. These experiments revealed that (+)- and (-)-ta[BP]G adducts on the transcribed strand of the DNA template block RNAPII in a sequence and stereochemistry-dependent manner; however, adducts on the non-transcribed strand do not block elongation significantly but may increase pausing at innate pause sites. In order to elucidate biologically influential differences between the (+)- and (-)-ta[BP]G structures, the DUPLEX program was used to carry out potential energy minimization searches at model transcription junctions. The lowest-energy minimum for the (+)-ta[BP]G adduct gives a structure in which the benzo[a]pyrenyl ring system resides in the minor groove of the heteroduplex region. In contrast, the lowest-energy minimum for a (-)-ta[BP]G adduct shows an orientation in which the benzo[a]pyrenyl group adopts a carcinogen/base-stacked conformation. These conformational preferences may contribute to the differential treatment of (+)- and (-)-ta[BP]G adducts by human RNAPII. In addition, while previous experiments showed that BPDE adducts cause T7RNAP to produce a ladder of truncated transcripts, RNAPII is blocked entirely at only one or two positions by the (+)- and (-)-ta[BP]G adducts, depending on sequence context. It is likely that these differences between the behaviors of T7RNAP and human RNAPII are a result of the structural characteristics of the enzymes' active sites, a hypothesis that is explored in light of their known crystal structures.