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Record Count: 16
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DESCRIPTION (provided by applicant):
Both genes and environmental toxins may act as risk factors for Parkinson's disease (PD), but their potential interplay remains poorly understood. This proposal seeks to investigate gene-environment interactions that are potentially relevant to PD using the model organism Drosophila melanogaster. Attention will be directed on genes and toxins associated with PD that affect mitochondrial dysfunction, protein ubiquitination and dopamine homeostasis. Over-expression of the Drosophila vesicular monoamine transporter (DVMAT) has been shown to protect against the neurotoxic effects of at least one mutant gene associated with PD (parkin), and at least one pesticide (rotenone) that selectively kills dopaminergic neurons. This study will therefore test the hypothesis that the neuroprotective effects of VMAT will extend to a different toxin, paraquat, thought to act by a different mechanism than rotenone, and whether this requires its localization to synaptic vesicles and if it is required at a specific time relative to neurotoxic insults. Studies here have shown that over-expression of mutant forms of parkin can cause dopaminergic cell degeneration in flies. If mutations in parkin function as a risk factor for PD, then additional environmental agents may enhance this risk. This study will test this hypothesis using rotenone, paraquat and other agents. Parallel experiments for gene-environment interactions will be performed with the PD related gene, pink1. Finally, already established pink mutant phenotypes will be used to screen for agents that rescue the neurotoxic effects of mitochondrial dysfunction.
DESCRIPTION (provided by applicant): The broad long-term objective of this proposal is to elucidate the role of the Fanconi Anemia (FA) pathway in protecting cells from the carcinogenic effects of Benzo[a]pyrene (B[a]P)-induced DNA damage. B[a]P is an abundant and ubiquitous environmental carcinogen that is metabolized intracellularly to generate Benzo[a]pyrene Di-hydrodiol-Epoxide (BPDE). BPDE-induced DNA damage triggers Rad18 (an E3 ligase)- dependent mono-ubiquitination of PCNA and recruitment of the specialized DNA polymerase Pol kappa to replication forks. In contrast with replicative DNA polymerases which stall at sites of DNA damage, Pol kappa can perform DNA synthesis using BPDE-adducted DNA as a template. Pol kappa-mediated TLS enables cells to tolerate the mutagenic and lethal effects of BPDE adducts, thereby contributing to genomic stability and tumor suppression. However, the molecular mechanism(s) of Pol kappa regulation are incompletely understood. Our preliminary studies suggest that the Fanconi Anemia (FA) proteins are intimately involved in Rad18 and Pol kappa- mediated TLS of B[a]P lesions. The Specific Aims of this proposal are: (1) To determine the molecular basis for BPDE-induced interaction between FANCD2 (the putative effector of the FA pathway) and Pol kappa. (2) To determine the mechanism by which Rad18 activates the FA pathway. (3) To test the interdependence of the FA and TLS pathways. Aim 1 will mutate ubiquitin binding motifs in Pol kappa and determine the effect of these mutations on FANCD2 association. Additionally, we will test the role of the Pol kappa- and FA pathway-interacting protein REV1 in mediating Pol kappa-FANCD2 interactions. Aim 2 will test whether Rad18 modifies FANCD2 directly or via regulation of upstream FA core complex components. Aim 3 will determine whether FANC- deficiency compromises TLS of BPDE lesions and conversely, whether defective TLS affects activation of the FA pathway by BPDE. Results of our studies will provide a novel paradigm for mechanisms by which the FA pathway is coordinated with TLS enzymes in response to environmental B[a]P (and possibly other genotoxins) to maintain genomic stability. Our studies might help identify individuals that are at high-risk for environmental B[a]P-induced disease. Moreover, our work could lead to novel drug targets for cancer therapy: Similar to B[a]P, many chemotherapies are genotoxic and activate checkpoint pathways. We have shown that Pol kappa- or Rad18-deficiency sensitizes cells to B[a]P-induced death. Potentially, small molecules that target FA components, Rad18, Pol kappa, or other TLS enzymes could sensitize cancer cells to killing by chemotherapeutic agents. PUBLIC HEALTH RELEVANCE: Benzo[a]pyrene (B[a]P) is an abundant environmental pollutant that damages DNA thereby causing permanent changes to the genetic code (termed 'mutations') that can result in cancer. Our results suggest that a group of cellular proteins termed the 'Fanconi Anemia' proteins (FANCs) play a role in preventing environmental B[a]P- induced mutations. The proposed experiments aim to understand the mechanisms by which FANCs help cells tolerate the effects of environmentally-induced DNA damage and protect against cancer.
DESCRIPTION (provided by applicant): The long-term objectives are to explain biochemically how human mismatch-repair (MMR) systems act in vivo to suppress induction of mutation by DNA lesions, yet promote lesion-triggered cell-cycle arrest and apoptosis. The focus here is on two representative classes of environmentally-induced mutagenic and carcinogenic lesions, UV photoproducts and polycyclicaromatic-hydrocarbon (PAH) adducts. The aim of the specific components of this exploratory work is to provide a well-defined basis for future studies-both expanded biochemical analyses and work with cultured cells and transgenic mice. Aim 1 is to analyze (ATP-sensitive) binding, by the (purified) human MMR-recognition proteins hMutS1 and hMutS2, of DNA containing defined cyclobutane-pyrimidine-dimer (CPD) photoproducts or PAH-purine adducts-opposite "matching" or "mismatching" nucleotides. The MMR targets here are, respectively, T[CPD]T/AG vs. T[CPD]T/AA in various contexts, and B[a]P-N2G:C vs. B[a]P-N2G:T and B[c]Ph-N6A:T vs. B[c]Ph-N6A:C. Targets bound twice as well as homoduplex DNA in electrophoretic-mobility-shift arrays will be used for further experiments. Aim 2 is to determine, using complete cell-free extracts, which Aim 1 lesion targets activate incision, by the essential MMR protein hMutL1, of model circular substrates (containing preexisting defined nicks in non-lesion strands) and concomitantly provoke 3'-5' excision along the shorter paths from substrate nicks to targets. Ability to activate MutL1 distinguishes "Class I" MMR targets (base- mispair prototype) from "Class II" (T[CPD]T/AG prototype) targets. Putative Class II targets will be tested, by surface-plasmon-resonance techniques, for abilities to form ternary complexes with (unactivated) hMutL1 and hMutS(1/2). Aim 3 is to test PAH-purine and the prototypic T[CPD]T/AG targets for the defining Class II property-activation of the MMR-associated hExoI protein for 5'-3' excision of model nicked-circular substrates by purified-protein mixtures and cell-free extracts. Aim 4 is to compare forward Hprt mutation induced in MMR-deficient vs. MMR-proficient cells in culture by B[a]P and B[C]Ph diol epoxides. Efficient MMR responses to DNA lesions can both reduce cancer risk and promote killing of tumor cells by some chemotherapeutic drugs. Environmental mutagens may pose unsuspected risks to individuals harboring one or (especially) multiple polymorphic partial MMR deficiencies. Additional partial deficiencies in legion-removal system may further. Future biochemical studies will analyze the efficiency and accuracy of DNA resynthesis past template lesions that have provoked 3'-5' (Aim 2) or 5'-3' (Aim 3) excision of (non-lesion) strands in extracts and recruitment (in the absence of excision) of signaling proteins in these extracts. Future genetic studies with various transgenic mice will examine effects of MMR-protein "knockouts" on specific mutation pathways induced by B[a]P and B[c]Ph diol epxidos. PUBLIC HEALTH RELEVANCE: This exploratory work will provide the basis for more later more extensive studies. These will help identify individuals at increased risk for cancer induced by environmental agents.
DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (pols) promote replication through DNA lesions. Humans possess four TLS pols that belong to the Y-family, Pols eta, iota, kappa, and Rev1, and another Pol, Pol zeta, that belongs to the B-family. The overall objectives of this proposal are to elucidate the biological roles of these pols in TLS in human cells, to determine whether they function in an error-free or mutagenic manner, and to identify the means by which a TLS Pol gains access to PCNA and thereby to the replication fork stalled at a DNA lesion site. These and related questions will be studied in the following Specific Aims. In Aim 1, the roles of TLS Pols in promoting replication through a diverse set of DNA lesions in human cells will be analyzed using an SV-40 based plasmid system that we have constructed. DNA lesions to be studied include UV photoproducts, and DNA adducts such as 1,N6-ethenodeoxyadenosine, 1,N2-propano-2'-deoxyguanosine, and (+) trans-dG-N2- benzo[a]pyrene diol epoxide, which result from cellular oxidative damage and from exposure to DNA damaging environmental carcinogens. In Aim 2, studies will be done to test the hypothesis that in human cells, Rev1 functions as a structural element for Pols eta, iota, and kappa. In Aim 3, the roles of TLS Pols in promoting replication through UV lesions and whether they do so in an error-free or mutagenic manner will be studied in the chromosomal cII transgene carried in a mouse embryonic fibroblast cell line. The contributions that cyclobutane pyrimidine dimers vs. (6-4) photoproducts make to UV mutations resulting from the action of different TLS Pols will be analyzed. In Aim 4, the model that, in addition to their binding to PCNA via their PIP domain, TLS Pols bind the ubiquitin moiety on PCNA via their ubiquitin binding domain, will be tested by genetic and biochemical studies of mutations in these domains of Pol eta. In Aim 5, biochemical studies will be undertaken to test the hypothesis that upon stalling at a DNA lesion site, the binding of the replicative pol to PCNA is attenuated as a result of PCNA ubiquitination, and that, in turn, promotes the access of the TLS pol to the primer-template junction via its binding to ubiquitinated PCNA (Ub-PCNA). Specifically, these studies will examine how Ub-PCNA promotes synthesis by Pol eta through a cis-syn TT dimer when a processively moving Pol delta has become stalled at the lesion site. By helping ensure the continuity of the replication fork, TLS Pols play an important role in the maintenance of genomic integrity. Furthermore, their proficient abilities for promoting error-free replication through a large variety of DNA adducts that result from cellular oxidative reactions and from exposure to chemical and environmental carcinogens will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. In fact, the inactivation of Pol eta in humans results in highly elevated levels of skin cancers. The proposed studies are highly relevant for cancer etiology as the results will reveal how human cells minimize the mutagenic and carcinogenic consequences of DNA lesions. PUBLIC HEALTH RELEVANCE: DNA lesions are generated in human cells from cellular oxidative reactions and from exposure to a variety of environmental pollutants and carcinogens. By promoting error-free replication through such DNA lesions, translesion synthesis DNA polymerases would play an important role in maintaining genome stability by keeping the rate of mutations and hence the incidence of cancers low. The proposed studies will elucidate the roles of human DNA polymerases in promoting replication through a variety of DNA lesions and they will examine the mechanisms of this process.
DESCRIPTION (provided by applicant): Hexavalent chromium (Cr(VI)) is a known human lung carcinogen. Millions of workers are exposed to Cr(VI) worldwide. The most recent paradigm proposes that the chromate compounds that are moderately water-soluble are more carcinogenic than the chromates that are either insoluble or water-soluble; however, it is not understood why this is so. Most of the data on how chromium damages DNA and causes DNA mutations has come from either the insoluble chromates or the water soluble chromates, not from the moderately soluble chromates that may be the most carcinogenic. The moderately soluble chromates are zinc chromate, strontium chromate and calcium chromate. Several crucial pieces of information are lacking regarding the activity of these moderately soluble chromates. No mutagenicity studies have been carried out with these compounds in human lung epithelial cells, which are the target cells for tumor formation. The types of mutations that these compounds cause have not been characterized in any cell line. The relative mutagenic potency of these chromates is not known. The extent to which these compounds dissolve outside of cells or enter cells as particulates is not known. The involvement of the zinc, strontium and calcium counterions in chromate toxicity is not known. The goals of the current proposal are to explain (1) how the moderately soluble Cr(VI) compounds enter cells and (2) if the mutations caused by the moderately soluble chromates differ from those caused by the soluble and insoluble chromates. The first aim of this proposal will apply the techniques of inductively coupled plasma mass spectrometry, laser scanning confocal microscopy, transmission electron microscopy, and scanning electron microscopy to determine how the moderately soluble chromates enter cells. The second aim of this proposal will measure and characterize mutations at the hypoxanthine (guanine) phosphoribosyl transferase (hprt) locus caused by the moderately soluble chromates in human lung epithelial cells and will compare mutation frequency and identity with mutations caused by the soluble and insoluble chromates. Data from these experiments will determine the relative mutagenic potency of these chromates, and will give insight into mechanisms of action, i.e., involvement of counterion and possible DNA lesions responsible for the mutations. Data from this proposal will provide mode of action information that will be necessary for thorough human risk assessment. The purpose of this work is to determine how chromium(VI) causes cancer. The goals of this proposal are to explain (1) how the moderately soluble chromium(VI) compounds enter cells and (2) if the mutations caused by the moderately soluble chromates differ from those caused by the less carcinogenic soluble and insoluble chromates. Understanding the mode of action of these chromates will provide a foundation for human risk assessment.
DESCRIPTION (provided by applicant): Many carcinogens become covalently attached to DNA and cause genotoxic damage due to polymerase blockage or nucleotide misincorporation. These events are controlled kinetically by the individual DNA polymerases. Continued studies are proposed on the interaction of a series of carcinogens, bound to oligonucleotides, with a set of both replicative and so-called translesion bypass polymerases, including viral (DNA polymerase T7-, HIV-1 reverse transcriptase) and bacterial (Dpo4) model polymerases as well as recombinant human polymerases (delta, eta, iota, kappa, REV1). Kinetic analyses will be done with these polymerases and the 22 DNA-carcinogen adducts available, varying in size from oxygen and methyl groups to large polycyclic hydrocarbons, identifying changes in rate-limiting events related to normal incorporation vs. misincorporation and blocking. The working hypothesis is that alternate conformations and inactive complexes are important, and some of these may also have rapid nucleotide dissociation kinetics. Another use of kinetic analysis will be to understand the chronology and coupling of the events involved in changing polymerases at DNA damage sites, i.e. between replicative and translesion polymerases. Roles of accessory proteins will be considered. Pre-steady-state kinetic spectroscopic approaches will also be used to better define events related to individual steps of polymerase catalysis, with fluorescence and circular dichroism changes being compared to measured rates of product formation. Work on the crystallography of carcinogen-adducted DNA with polymerases (HIV-1 reverse transcriptase, Dpo4) is in progress and will be expanded, with the goal of linking temporal events with specific structural changes. The general working hypothesis is that normal incorporation, misincorporation, pausing, and blockage represent a continuum of events related to fits and rates of reactions involving polymerase-DNA-dNTP ternary complexes, and that these can be understood in quantitative terms (rate constants and structures). The overall goal is understanding molecular mechanisms of mutagenesis and relevance to chemical carcinogenesis.
DESCRIPTION (provided by applicant): DMA repair and damage tolerance processes are absolutely critical to preserving human health following exposure to many different agents. The long term goal of this research is to develop a detailed integrated understanding of the molecular mechanisms responsible for environmental mutagenesis in eukaryotes. In molecular processes that are both complex and elaborately controlled, mutations are introduced when specialized translesion synthesis (TLS) DNA polymerases copy over DNA damage caused by environmental agents. The proposed research places a special emphasis on Rev1, which by virtue of acting both as a scaffold for other TLS DNA polymerases and as a polymerase itself, lies at the root of eukaryotic mutagenesis. We will follow up on our unanticipated finding that S. cerevisiae Rev1 is expressed 50 fold higher during G2/M than in G1 and most of S phase by investigating the basis of its cell cycle control," which is predominantly posttranscriptional; promising candidate regulatory genes such as UMP1 and CDC7 will be tested, genetic screens for new regulatory genes will be carried out, and experiments will be conducted to determine the importance Of this control: Using both biochemical and genetic approaches, we will continue to investigate the structural and functional basis of Rev1 interactions, focusing particularly on the interactions of the Rev1 C-terminal domain, its BRCT domain, and its ubiquitin-binding motifs. We will investigate the functional importance of the Rev1 polymerase activity by following up on our recent observations suggesting that Rev1 may have a class of cognate lesions it replicates particularly well. We will investigate the function and regulation of S. pombe DinB and its relationship to Rev1. We will develop a high-throughput assay, based on disruption of the Rev1-Rev7 interaction, that will allow screening for inhibitors of eukaryotic environmental mutagenesis. The proposed research will make a highly significant contribution to basic science by elucidating the still poorly understood eukaryotic translesion synthesis mechanisms responsible for most mutations. These mutations contribute to aging, cancer, and various human diseases. Identification of additional genes that play roles in these mutagenic processes will help make it possible to address the question of why only some people develop disease when exposed to an environmental toxin. Small molecule inhibitors of TLS could lead to novel "anti-mutagenesis" drugs with multiple applications to human health.
DESCRIPTION (provided by applicant): The importance of somatic mutation in the genesis of cancer and other diseases is undisputed. However, the extent to which loss of heterozygosity (LOH), as a consequence of mitotic recombination, contributes to the frequency of spontaneous mutation has been under-appreciated. Using a mouse model that is heterozygous at Aprt we have previously reported that in vivo spontaneous mutation frequencies at Aprt can approach 10-4 and that up to 80 percent of these events can be due to mitotic recombination. The global mutation frequencies, in fact, are even higher given that all loci between the point of crossover and the reporter locus are also affected, and that mitotic recombination can occur between all autosome homologs. The goals of this program are to further exploit our Aprt heterozygous model and to develop additional Aprt-derived models to ask questions regarding local events at sites of double strand breaks, and to determine whether different pathways to mutation/repair are preferred in different isogenic cell types. An emphasis will be placed on embryonic stem (ES) cells and adult stem cells since they have requirements beyond those of somatic cells for preserving the integrity of their genomes. We have already reported that ES cells suppress mutation and mitotic recombination by as much as 100 fold compared with isogenic mouse embryo fibroblasts (MEFs), one mechanism to protect their genomes. We and others have also shown that ES cells are hypersensitive to DNA damage, and that they lack a G1 checkpoint, presumably facilitating death and removal of cells that have acquired a mutational burden. This represents a second mechanism by which ES cells protect their genomes. We have identified the signaling pathway that is compromised in ES cells after DNA damage, reconstituted it, and shown that reestablishment of a G1 arrest after challenge protects the cells from apoptosis. We wish to ask whether adult stem/progenitor cells behave more like somatic cells or ES cells with respect to cell cycle regulatory mechanisms and mechanisms to suppress mutation. The role of adult stem cells in the genesis of tumors is an important question that is not yet resolved. We will use our Aprt null mice and intestinal epithelium to ask whether mutations in the mouse gut arise exclusively in the crypt base, site of the progenitor cells or whether they also arise elsewhere within the crypt. This experiment should provide evidence for or against the "top down" model of colon cancer that has been proposed.
DESCRIPTION (provided by applicant): The long term goal of this research program is to uncover and understand mutational pathways, the DNA repair processes or avoidance mechanisms that counteract these pathways and the consequences of defects in these systems. Our basic approach has often involved characterizing "mutator strains" that have higher mutation rates than wild-type, and then determining the affected pathway or repair system. We have developed new approaches for the detection of mutators, and this has allowed us to define several new mutational pathways, including one that results from the overexpression of the EmrR repressor of a multi-drug resistance efflux pump. Overexpressing a common repressor is one way to detect mutational pathways that back each other up and thus require two knockouts to produce a mutator phenotype. We hypothesize that this system acts to pump out mutagenic products of metabolism and represents the cell's first line of defense against mutagenesis. This proposal seeks to test this idea by further charactering this novel system, and also to characterize other new mutational pathways we have discovered. For instance, the use of metagenomic libraries has revealed that multiple copies of extensively repeated sequences results in global genomic instability. This will be further investigated. We will also utilize the recently completed E. coli knockout collection, consisting of close to 4,000 strains, each carrying an in-frame deletion of one of the orfs in the E. coli genome, together with our own tools to screen for new mutational pathways. We will use gene fusions to study the regulation of different repair genes and to characterize the action of new mutators, and will use several pathogen or extremophile genomes (Bordetella pertussis, Campylobacterjejeuni, and Deinococcus radiodurans) as a source for genes that might provoke mutator phenotypes in E. coli, and also that would complement different E. coli repair defects. We will also construct a system for studying mutagenesis in a pathogen such as B. pertussis.
DESCRIPTION (provided by applicant): The potent environmental carcinogen benzo[a]pyrene (B[a]P) is metabolically activated in cells to (+)-anti-B[a]PDE, which forms 1 primary adduct: [+ta]-B[a]P-N2-dG. [+ta]-B[a]P-N2-dG induces different mutational patterns depending on sequence context (e.g., >95% G->T vs. 95% G->A in 5'-TGC vs. 5'-AGA sequences). Evidence suggests that these different mutations arise from different adduct conformations (as influenced by sequence context), when bypassed by different DNA polymerases. For [+ta]-B[a]P-N2-dG, we showed that E. coli DNA Pol V is involved in dATP bypass (G->T mutations), while dCTP insertion (no mutation) involves Pols IV and V. With the mirror image adduct [-ta]-B[a]P-N2-dG, Pol V does dATP insertion, while Pol IV alone is required for dCTP bypass. Literature findings suggest that in general DNA Pol V has 2 modes of adduct bypass: (1) correct dNTP insertion, and (2) default dATP insertion. Understanding the mechanism of correct vs. mutagenic insertion is hampered by no X-ray structure for UmuC, which is the polymerase subunit of DNA Pol V. Using homology modeling, we constructed a UmuC model, which revealed active site amino acids potentially involved in dictating dNTP insertion. Active site amino acids were changed. In cells we showed that mutant-UmuCs could increase (up to ~10-fold), or decrease (~5-fold) dATP insertion compared to wt-UmuC. The goal of this project is to understand what amino acid residues define correct (dCTP) vs. incorrect (dATP) insertion for Pols IV and V and how these pathways are controlled by the cell. Studies in cells and in vitro with mutant and wild type Pols IV and V are proposed. Literature findings show that Pol IV is equivalent to human Pol k, while Pol V is equivalent to human Pol h. Aim 1. Establish the roles of Pol IV vs. Pol V in cells; i.e., which does insertion vs. extension. Aim 2: Determine what amino acids in Pols IV and V control correct (dCTP) vs incorrect (dATP) insertion; e.g., why does Pol IV do correct (dCTP) insertion, while Pol V does incorrect (dATP) insertion, with [-ta]-B[a]P-N2-dG. Aim 3: Determine what lesion-bypass Pols are involved in G->A mutagenesis (dTTP insertion).
Crisp Terms/Key Words: enzyme activity, tissue /cell culture, bacterial protein, protein sequence, protein biosynthesis, DNA directed DNA polymerase, deoxyribonucleoside triphosphate, gene mutation, mutagen testing, mutagen, benzopyrene, adduct, Escherichia coli, aminoacid
DESCRIPTION (provided by applicant): The broad objectives of this grant are to investigate biochemical mechanisms by which damaged DNA is copied by the cell's replication and repair enzymes, focusing on proteins that are induced in response to DNA damage. Damage-induced DNA repair occurs in both procaryotic and eucaryotic organisms. In Escherichia coli, response to DNA damage is orchestrated by an operon, the "SOS regulon", containing more than 40 different proteins under negative control of a repressor protein, LexA, and a multifunctional protein, RecA. In E. coli, and in animal cells, damage-induced DNA repair can often be aberrant. There is a reduction in fidelity that enables replication to continue past blocking DNA damage sites. The primary goal of this proposal is to elucidate the biochemical basis for SOS-induced error-prone repair in E. coli. Such repair depends on RecA protein interacting with a mutagenic UmuD'2C protein complex, which we showed to be a new DNA polymerase, E. coli pol V. The discovery of this new polymerase provided the impetus for the "explosive" growth based on subsequent discoveries of fundamentally important error- prone eukaryotic DNA repair polymerases involved, for example, in avoiding skin cancer and in generating antibody diversity. There are numerous types of damage occurring in DNA when cells are exposed to chemicals, drugs or radiation. To study the biochemical basis of error-prone repair in vitro, we have chosen to focus primarily on copying site-directed biologically relevant lesions that occur spontaneously or from exogenous DNA damage. DNA template lesions often present a strong block to DNA replication. When replication past a lesion does occur, it can generally cause a mutation targeted to the DNA damage site. In this proposal, we will focus on the key biochemical interactions responsible for error-prone translesion DNA synthesis, involving E. coli DNA polymerase V, RecA protein, and polymerase processivity clamp proteins. We intend to determine the mechanisms governing targeting of repair polymerases to damaged DNA and mechanisms of trafficking between polymerases, to exchange high fidelity replication polymerase blocked at a site of DNA damage with low fidelity repair polymerases that can relieve the blockage at the expense of generating mutations. During the previous grant period, we discovered an unprecedented mechanism in DNA replication by which E. coli DNA polymerase V is unable to copy damaged or undamaged DNA unless activated in trans by RecA bound to ssDNA not being copied by pol V. A central theme of this proposal is to establish the mechanistic basis for RecA- ssDNA transactivation of pol V. The data generated in the proposed experiments will have major biological impact in exploring the principles of how damaged DNA is copied.
Project Narrative: In organisms ranging from bacteria to humans, almost all mutations are deleterious, serving as a root cause of numerous diseases including cancer. Ironically, however, it has recently been found that there are error-prone DNA repair pathways that are beneficial, indeed essential, in providing immunological diversity, general fitness and avoidance of cell death, and even protecting against some human diseases, for example skin cancer. The proposed research explores the biochemical basis governing the ability of error-prone DNA polymerases to copy damaged DNA that would otherwise cause a cessation of chromosome replication leading to cell death.
DESCRIPTION (provided by applicant): The biological consequences of cytoplasmic damage are largely unknown. The prevailing dogma considered the genotoxic effects of environmental carcinogens such as polycyclic aromatic hydrocarbons and radon alpha particles as being due mostly to direct damage to the nucleus. Using a precision charged particle microbeam and dual fluorochrome dyes to locate nucleus and cellular cytoplasm respectively, thereby avoiding inadvertent traversal of nuclei, the applicant has shown previously that cytoplasmic irradiation is, in fact, mutagenic at the CD59 locus of human-hamster hybrid (AL) cells while inflicting minimal cytotoxicity. Furthermore, preliminary evidence suggests that reactive oxygen species mediate this process. This raised the following questions: What types of oxyradicals are involved and what are their origins? Does this radical generating process involve mitochondrial damage? Do the mutations induced by targeted cytoplasmic irradiation occur in human bronchial epithelial cells (target tissues of environmental radon) as well? And finally, can cytoplasmic irradiation induce bystander mutagenic effect in mammalian cells in a manner similar to what the applicant has recently demonstrated with nuclear traversal of the AL cells. To address these issues, a series of 5 specific aims are proposed to address the 4 testable hypotheses. Mutations will be scored at the CD59 locus of the AL cells and at the HPRT locus in primary human bronchial epithelial cells. The proposed studies will help to address the mechanisms of how cytoplasmic irradiation results in a genetic event in the nucleus. Together with the bystander mutagenic effect, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells.
DESCRIPTION (provided by applicant): The frequent consumption of meats cooked well-done leads to an increased risk for colon and breast cancer; however, the etiological agents responsible for this risk remain to be elucidated. The Report on Carcinogens, Eleventh Edition, National Toxicology Program, concluded that heterocyclic aromatic amines (HAAs), which arise in grilled meats, are reasonably anticipated to be human carcinogens. Many epidemiological studies have implicated HAAs as specific etiological agents in these cancers. However, the reported associations of dietary factors and genetic polymorphism data can not confirm the relationships between specific chemical exposures and carcinogenesis. Moreover, the characterized HAAs account for less than 30% of the mutagenicity attributed to this class of chemicals in well-done grilled meats and other uncharacterized HAAs are present. Recently, we discovered 2-amino-1-7-dimethylimidazo[4,5-g]quinoxaline (7-MeIgQx), an isomer of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), a potent animal carcinogen. 7-MeIgQx, is the most mass-abundant, genotoxic HAA formed in grilled beef; however, its carcinogenic potential is unknown. Our long-term goal is to assess the cancer risk posed by HAAs, by establishing chemical markers that may distinguish individuals at different levels of risk. Given the high concentrations of 7-MeIgQx formed in cooked beef in relationship to 8-MeIQx or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), two HAAs that are believed to contribute to dietary-related cancers, we hypothesize that 7-MeIgQx is a significant contributor to the overall genotoxic burden and cancer risk posed by HAAs. The objective of this application is to provide preliminary toxicological data on the capacity of 7-MeIgQx to undergo bioactivation in vitro and bind to DNA in vivo in the rat. We will use highly sensitive accelerator mass spectrometry methods to measure DNA adduct yields of 7-MeIgQx in vivo and compare the yields to those of 8-MeIQx. The rationale is that data generated from these pilot studies will provide a preliminary assessment of the carcinogenic potential of 7-MeIgQx in comparison to 8-MeIQx, based on the carcinogen binding index. This proposed research is relevant to NIH's mission on public health: specific aims 1 and 2 will establish toxicological data on the genotoxic potential of 7-MeIgQx in relationship to its carcinogenic isomer, 8-MeIQx. The proposed DNA binding study in vivo of a newly discovered HAA is an innovative approach to provide a rapid, provisional assessment of the carcinogenic potential of 7-MeIgQx, which arises cooked beef in greater quantities than 8-MeIQx and PhIP. This discovery is highly significant since metabolites and DNA adducts of 7- MeIgQx could serve as biomarkers of exposure and risk assessment to this class of dietary carcinogens. PUBLIC HEALTH RELEVANCE: The proposed study will provide a provisional estimate of the carcinogenic potential of a newly discovered heterocyclic aromatic amine (HAA), 2-amino-1,7-dimethylimidazo[4,5-g]quinoxaline (7-MeIgQx). HAAs are believed to be human carcinogens and 7-MeIgQx is the most mass-abundant HAA formed in cooked meat. The research will allow us to determine the health risk of 7-MeIgQx in relationship to other HAAs present in the diet.
DESCRIPTION (provided by applicant): A major function of telomeres is to protect the ends of chromosomes and prevent chromosome fusion. Chromosome fusion can result in chromosome instability through breakage/fusion/bridge (B/F/B) cycles, which occur when chromosomes repeatedly break and fuse with each cell division. B/F/B cycles can be prevented or terminated by the addition of telomeres to the ends of broken chromosomes, which results in the formation of terminal deletions. In Tetrahymena and yeast, telomeres are added on to the ends of broken chromosomes by telomerase, termed chromosome healing. Chromosome rearrangements associated with telomere loss and B/F/B cycles have been found in a variety of human genetic diseases, and are thought to play an important role in the chromosome instability associated with cancer. We have established an assay that utilizes selectable marker genes located adjacent to a telomere to monitor the consequences of telomere loss in mammalian cells. Telomere loss is induced through the introduction of a double-strand break at an I-Scel site adjacent to the telomere following the expression of the I-Scel endonuclease. Most mouse embryonic stem cells that lose a telomere have telomeres added directly on at the I-Scel site; however, sister chromatid fusion and chromosome instability involving B/F/B cycles is observed in cells that do not add a telomere on to the end of the broken chromosome. The present proposal will use this system to address the consequences of telomere loss in mammalian cells. Specific Aim 1 will investigate the role of telomerase and the Pif1 helicase in chromosome healing. Pif1 mutants in yeast have a 600-fold higher incidence of chromosome healing, and as a result, Pif1 has been proposed to negatively regulate chromosome healing to prevent terminal deletions. These experiments will test the hypotheses that telomerase and Pifl are important in chromosome healing in mammalian cells and that chromosome healing prevents B/F/B cycles. Specific Aim 2 will utilize cell lines containing mutations in DNA-PKcs or NBS1, to address the role of nonhomologous end joining and the Mre11/Rad50/Nbs1 complex in telomere maintenance, chromosome healing and chromosome fusion. Specific Aim 3 will test the hypothesis that chromosome instability due to telomere loss promotes carcinogenesis by expressing I-Scel in vivo and monitoring preneoplastic changes resulting from the amplification/rearrangement of the c-Myc gene on the chromosome 15 containing a telomeric I-Scel site.
DESCRIPTION (provided by applicant): 1,3-butadiene (BD) is a known carcinogen. However, the DNA adducts responsible for mutations remain unknown. The overall goals of the proposed research are to examine the molecular dose of previously unexplored DNA adducts in rodents exposed to BD and 3-butene-1, 2-diol (BD-diol), comparing the data with mutation frequencies and mutational spectra to determine if a particular adduct could be used as a quantitative indicator of mutagenesis, and to evaluate effects of exposure on gene expression. The first hypothesis to be tested is that hydroxymethylvinyl ketone (HMVK) is formed in vivo during exposure to BD and BD-diol in a sex, species, and exposure concentration dependent manner resulting in important differences in mutagenicity. The second hypothesis is that promutagenic N1 adenine adducts convert to the more stable inosine adducts which are poorly repaired and accumulate during chronic exposure to BD. Several specific aims will be accomplished while addressing these hypotheses. Specific Aim 1 is to examine the formation of potentially mutagenic DNA adducts (specifically 1, N2-propanodeoxyguanosine) by HMVK in vivo. The second aim is to determine the utility of the N-terminal valine adduct of HMVK (HMVK-Val) as a biomarker of HMVK formation by BD and BD-diol. Specific Aim 3 is to develop methods for detecting N1- inosine, N1 - and N6 adenine adducts derived from BD metabolites in vivo. Specific Aim 4 is to determine the mutagenic responses induced by BD exposures and characterize the impact of BD-diol derived metabolites on the spectra of mutations induced by BD exposure in the B6C3F1 mouse and F344 rat to identify which adducts studied in Aims 1 and 3 are quantitative indicators of mutagenesis. Specific Aim 5 will examine the effects of exposure to BD and BD-diol on gene expression and DNA repair pathways. Collectively, these experiments have been designed to look at adduct formation, DNA repair, mutagenicity, and genomic alterations in rodents exposed BD and BD-diol, as well as the impact of glutathione depletion and DNA repair deficiency. Finally, HMVK-Val adducts will be measured in samples from BD exposed humans. Our research will identify critical metabolites and adducts that are responsible for BD mutagenicity, as well as develop biomarkers suitable for future molecular epidemiology studies. Ultimately these data will improve our understanding of critical mechanisms of toxicity and ability to accurately assess the risk of BD to humans.
Crisp Terms/Key Words: environmental exposure, clinical research, DNA damage, technology /technique development, inosine, DNA repair, chemical carcinogen, ketone, human tissue, gene mutation, mutagen, environmental contamination, diene, adduct, biomarker, laboratory rat, laboratory mouse
DESCRIPTION (provided by applicant): Rev1 is unique among DNA polymerases in that the protein itself rather than the DNA template determines the specificity for both the templating and the incoming nucleotide. The Rev1 crystal structure that we have solved suggests an elegant mechanism by which this polymerase could promote proficient and error-free replication through a large variety of N2-adducted guanines that result from endogenous oxidative damage and from exposure to a number of widespread DNA damaging chemical and environmental carcinogens such as butadiene epoxides and anti-benzo[a]pyrene diol epoxides. Such a role for yeast Rev1 will be examined using a combined biochemical, genetic, and structural approach. In Aim 1, key amino acid residues involved in the pairing with the incoming dCTP and in the eviction and stabilization of templating G will be mutated and their effects on nucleotide incorporation specificity and catalytic efficiency determined. In Aim 2, the conformational changes that occur in Rev1 upon DNA binding and upon dNTP binding will be analyzed through crystal structures of the Rev1 apoenzyme and Rev1.DNA binary complex and their comparison to the structure of Rev1.DNA.dCTP ternary complex. In Aim 3, biochemical studies will be undertaken to test the hypothesis that a major role of the Rev1 DNA synthetic activity is to promote efficient and error-free replication through various N2adducts of guanine that sterically impinge upon the minor groove, and which result from cellular oxidative damage or from exposure to DNA damaging environmental carcinogens. Also as part of this aim, we will examine the means by which complex formation between Rev1 and the extender polymerase coordinates the nucleotide insertion and the subsequent extension steps in the bypass of these adducts. As a complement to these biochemical studies, in Aim 4, crystal structures of Rev1 with DNAs containing a variety of N2 guanine adducts will be determined, as well as the structure of Rev1 with an abasic lesion. In Aim 5, genetic studies will be done to establish the requirement of the Rev1 DNA synthetic activity in promoting error-free replication through the various N2-adducts of guanine in yeast cells. Rev1 as well as the other DNA repair proteins are highly conserved between yeast and humans. The proficient and accurate ability of Rev1 for promoting replication through the large variety of DNA adducts that form at the N2 of guanine will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The results of this study are highly relevant for cancer biology and etiology, as error-free replication through DNA lesions provides for an important means of cancer prevention. PUBLIC HEALTH RELEVANCE: DNA lesions generated from cellular oxidative damage and from exposure to environmental pollutants affect the stability and integrity of genomic DNA. Error-free replication through such lesions reduces their adverse impact by keeping the rate of mutations low and by reducing the incidence of cancer formation. The proposed studies will examine the role of Rev1 DNA polymerase in promoting error-free replication through DNA lesions.
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