How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene.

To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N(2)-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N(2)-dG.

A method to accurately quantitate intensities of (32)P-DNA bands when multiple bands appear in a single lane of a gel is used to study dNTP insertion opposite a benzo[a]pyrene-dG adduct by Sulfolobus DNA polymerases Dpo4 and Dbh.

Quantitating relative (32)P-band intensity in gels is desired, e.g., to study primer-extension kinetics of DNA polymerases (DNAPs).

Structural model of the Y-Family DNA polymerase V/RecA mutasome.

To synthesize past DNA damaged by chemicals or radiation, cells have lesion bypass DNA polymerases (DNAPs), most of which are in the Y-Family. One class of Y-Family DNAPs includes DNAP η in eukaryotes and DNAP V in bacteria, which have low fidelity when replicating undamaged DNA. In Escherchia coli, DNAP V is carefully regulated to insure it is active for lesion bypass only, and one mode of regulation involves interaction of the polymerase subunit (UmuC) and two regulatory subunits (UmuD') with a RecA-filament bound to ss-DNA.

Architecture of y-family DNA polymerases relevant to translesion DNA synthesis as revealed in structural and molecular modeling studies.

DNA adducts, which block replicative DNA polymerases (DNAPs), are often bypassed by lesion-bypass DNAPs, which are mostly in the Y-Family. Y-Family DNAPs can do non-mutagenic or mutagenic dNTP insertion, and understanding this difference is important, because mutations transform normal into tumorigenic cells. Y-Family DNAP architecture that dictates mechanism, as revealed in structural and modeling studies, is considered.

Amino acid architecture that influences dNTP insertion efficiency in Y-family DNA polymerase V of E. coli.

Y-family DNA polymerases (DNAPs) are often required in cells to synthesize past DNA-containing lesions, such as [+ta]-B[a]P-N(2)-dG, which is the major adduct of the potent mutagen/carcinogen benzo[a]pyrene. The current model for the non-mutagenic pathway in Escherichia coli involves DNAP IV inserting deoxycytidine triphosphate opposite [+ta]-B[a]P-N(2)-dG and DNAP V doing the next step(s), extension. We are investigating what structural differences in these related Y-family DNAPs dictate their functional differences.

Y-Family DNA polymerases may use two different dNTP shapes for insertion: a hypothesis and its implications.

Chemicals and radiation can damage DNA leading to the formation of adducts/lesions, which - if not removed by DNA repair pathways - usually block replicative DNA polymerases (DNAPs). To overcome such potentially lethal blockage, cells have lesion bypass DNAPs, which are often in the Y-Family and include several classes. One class includes human DNAP kappa and E.

Environmental and occupational causes of cancer: new evidence 2005-2007.

What do we currently know about the occupational and environmental causes of cancer? As of 2007, the International Agency for Research on Cancer (IARC) identified 415 known or suspected carcinogens. Cancer arises through an extremely complicated web of multiple causes, and we will likely never know the full range of agents or combinations of agents. We do know that preventing exposure to individual carcinogens prevents the disease.

Molecular modeling benzo[a]pyrene N2-dG adducts in the two overlapping active sites of the Y-family DNA polymerase Dpo4.

The potent, ubiquitous environmental mutagen/carcinogen benzo[a]pyrene (B[a]P) induces a single major adduct [+ta]-B[a]P-N2-dG, whose bypass in most cases results in either no mutation (dCTP insertion) or a G-->T mutation (dATP insertion). Translesion synthesis (TLS) of [+ta]-B[a]P-N2-dG generally requires DNA polymerases (DNAPs) in the Y-family, which exist in cells to bypass DNA damage caused by chemicals and radiation. A molecular dynamics (MD) study is described with dCTP opposite [+ta]-B[a]P-N2-dG in Dpo4, which is the best studied Y-family DNAP from a structural point of view.

Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells.

Fapy.dG and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5'-TGT and 5'-TGA sequence contexts.

Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g., G-to-T, G-to-A, -1 frameshifts, etc.

Homology modeling of four Y-family, lesion-bypass DNA polymerases: the case that E. coli Pol IV and human Pol kappa are orthologs, and E. coli Pol V and human Pol eta are orthologs.

Y-family DNA polymerases (DNAPs) are a superfamily of evolutionarily related proteins that exist in cells to bypass DNA damage caused by both radiation and chemicals. Cells have multiple Y-family DNAPs, presumably to conduct translesion synthesis (TLS) on DNA lesions of varying structure and conformation. The potent, ubiquitous environmental mutagen/carcinogen benzo[a]pyrene (B[a]P) induces all classes of mutations with G-->T base substitutions predominating.

Mutagenesis studies with four stereoisomeric N2-dG benzo[a]pyrene adducts in the identical 5'-CGC sequence used in NMR studies: G->T mutations dominate in each case.

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) and a potent mutagen/carcinogen found ubiquitously in the environment. B[a]P is primarily metabolized to diol epoxides, which react principally at N2-dG in DNA. B[a]P-N2-dG adducts have been shown to induce a variety of mutations, notably G-->T, G-->A, G-->C and -1 frameshifts.

Free-energy perturbation methods to study structure and energetics of DNA adducts: results for the major N2-dG adduct of benzo[a]pyrene in two conformations and different sequence contexts.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is activated to (+)-anti-B[a]PDE, which induces a variety of mutations (e.g., G --> T, G --> A, etc.

Mutagenesis studies of the major benzo[a]pyrene N2-dG adduct in a 5'-TG versus a 5'-UG sequence: removal of the methyl group causes a modest decrease in the [G->T/G->A] mutational ratio.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations primarily at the G:C base pairs (e.g. GC-->TA, GC-->AT, etc.

A role for DNA polymerase V in G --> T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT sequence in E. coli.

Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC --> TA, GC --> AT, etc.

Molecular modeling of the major benzo[a]pyrene N2-dG adduct in cases where mutagenesis results are known in double stranded DNA.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC-->TA, GC-->AT, etc.

Molecular modeling of four stereoisomers of the major B[a]PDE adduct (at N(2)-dG) in five cases where the structure is known from NMR studies: molecular modeling is consistent with NMR results.

The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which is known to induce a variety of mutations (e.g., GC --> TA, GC --> AT, etc.

The precautionary principle in environmental science.

Environmental scientists play a key role in society's responses to environmental problems, and many of the studies they perform are intended ultimately to affect policy. The precautionary principle, proposed as a new guideline in environmental decision making, has four central components: taking preventive action in the face of uncertainty; shifting the burden of proof to the proponents of an activity; exploring a wide range of alternatives to possibly harmful actions; and increasing public participation in decision making. In this paper we examine the implications of the precautionary principle for environmental scientists, whose work often involves studying highly complex, poorly understood systems, while at the same time facing conflicting pressures from those who seek to balance economic growth and environmental protection.

Factors that influence the mutagenic patterns of DNA adducts from chemical carcinogens.

Carcinogens are generally mutagens, which is understandable given that tumor cells grow uncontrollably because they have mutations in critical genes involved in growth control. Carcinogens often induce a complex pattern of mutations (e.g.

Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N(2)-dG) of the potent environmental carcinogen, benzo[a]pyrene.

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question - what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g.

DNA guanine-guanine crosslinking sequence specificity of isophosphoramide mustard, the alkylating metabolite of the clinical antitumor agent ifosfamide.

Purpose: The purpose of this investigation was to determine the base sequence specificity of isophosphoramide mustard (IPM), the alkylating metabolite of ifosfamide, by crosslinking of designed DNA oligomers in comparison with the clinical alkylating agents mechlorethamine (ME) (nitrogen mustard) and phosphoramide mustard (PM), the alkylating metabolite of cyclophosphamide.

Methods: IPM, as well as PM and ME were each reacted with three dodecameric duplexes, which were designed to detect interstrand crosslinking between guanines in 5'-GC-3' (I), 5'-GNC-3' (II) or 5'-GNNC-3' (III) sequences (N = A or T).

Results: All three agents preferentially react with 5'-GNC-3' target sequences.

DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli.

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6.

The major, N2-dG adduct of (+)-anti-B[a]PDE induces G-->A mutations in a 5'-AGA-3' sequence context.

Previously, in a random mutagenesis study, the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] was shown to induce a complex mutational spectrum in the supF gene of an Escherichia coli plasmid, which included insertions, deletions and base substitution mutations, notably a significant fraction of GC-->TA, GC-->AT and GC-->CG mutations. At some sites, a single type of mutation dominated and to understand individual mutagenic pathways these sites were chosen for study by site-specific means to determine whether the major adduct, [+ta]-B[a]P-N2-dG, was responsible. [+ta]-B[a]P-N2-dG was shown to induce approximately 95% G-->T mutations in a 5'-TGC-3' sequence context and approximately 80% G-->A mutations in a 5'-CGT-3' sequence context.