Activity of DNA polymerase κ across the genome in human fibroblasts.

DNA polymerase κ (Polκ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Polκ utilizing a Polκ-specific substrate, -(4-ethynylbenzyl)-2'-deoxyguanosine (EBndG). After shearing of the DNA into 200 to 500 bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)-catalyzed alkyne-azide cycloaddition reaction and isolated with streptavidin beads.

Active Site Interactions Impact Phosphoryl Transfer during Replication of Damaged and Undamaged DNA by Escherichia coli DNA Polymerase I.

Replicative DNA polymerases are able to discriminate between very similar substrates with high accuracy. One mechanism by which E. coli DNA polymerase I checks for Watson-Crick geometry is through a hydrogen bonding fork between Arg668 and the incoming dNTP and the minor groove of the primer terminus.

Mutagenic Replication of N-Deoxyguanosine Benzo[a]pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus solfataricus DNA Polymerase IV.

Benzo[a]pyrene, a potent human carcinogen, is metabolized in vivo to a diol epoxide that reacts with the N-position of guanine to produce N-BP-dG adducts. These adducts are mutagenic causing G to T transversions. These adducts block replicative polymerases but can be bypassed by the Y-family translesion synthesis polymerases.

N -Substituted 2'-Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ.

N -Alkyl-2'-deoxyguanosine triphosphate (N -alkyl-dGTP) derivatives with methyl, butyl, benzyl, or 4-ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N -Benzyl-dGTP was equal to dGTP as a substrate for DNA polymerase κ (pol κ), but was a poor substrate for pols β, δ, η, ι, or ν. In vivo reactivity was evaluated through incubation of N -4-ethynylbenzyl-dG with wild-type and pol κ deficient mouse embryonic fibroblasts.

Honokiol Inhibits DNA Polymerases β and λ and Increases Bleomycin Sensitivity of Human Cancer Cells.

A major concept to sensitize cancer cells to DNA damaging agents is by inhibiting proteins in the DNA repair pathways. X-family DNA polymerases play critical roles in both base excision repair (BER) and nonhomologous end joining (NHEJ). In this study, we examined the effectiveness of honokiol to inhibit human DNA polymerase β (pol β), which is involved in BER, and DNA polymerase λ (pol λ), which is involved in NHEJ.

DNA Polymerase ν Rapidly Bypasses O-Methyl-dG but Not O-[4-(3-Pyridyl)-4-oxobutyl-dG and O-Alkyl-dTs.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco carcinogen that forms mutagenic DNA adducts including O-methyl-2'-deoxyguanosine (O-Me-dG), O-[4-(3-pyridyl)-4-oxobut-1-yl]-dG (O-POB-dG), O-methylthymidine (O-Me-dT), and O-POB-dT. We evaluated the ability of human DNA polymerase ν to bypass this damage to evaluate the structural constraints on substrates for pol ν and to evaluate if there is kinetic evidence suggesting the in vivo activity of pol ν on tobacco-induced DNA damage. Presteady-state kinetic analysis has indicated that O-Me-dG is a good substrate for pol ν, while O-POB-dG and the O-alkyl-dT adducts are poor substrates for pol ν.

DNA Polymerases η and ζ Combine to Bypass O(2)-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are important human carcinogens in tobacco products. They are metabolized to produce a variety 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts including O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O(2)-POB-dT and O(2)-Me-dT by purified human DNA polymerases η, κ, ι, and yeast polymerase ζ in vitro.

Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent human carcinogen. Metabolic activation of NNK generates a number of DNA adducts including O(2)-methylthymidine (O(2)-Me-dT) and O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT). To investigate the biological effects of these O(2)-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O(2)-Me-dT or O(2)-POB-dT in human embryonic kidney 293T (HEK293T) cells.

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency.

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases.

Low fidelity bypass of O(2)-(3-pyridyl)-4-oxobutylthymine, the most persistent bulky adduct produced by the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by model DNA polymerases.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most important human carcinogens. It is metabolized to produce a variety of methyl and 4-(3-pyridyl)-4-oxo-butyl (POB) DNA adducts. A potentially important POB adduct is O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT) because it is the most abundant POB adduct in NNK-treated rodents.

In vitro epsilon RNA-dependent protein priming activity of human hepatitis B virus polymerase.

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a pregenomic RNA (pgRNA) by using a multifunctional polymerase (HP). A critical function of HP is its specific recognition of a viral RNA signal termed ε (Hε) located on pgRNA, which is required for specific packaging of pgRNA into viral nucleocapsids and initiation of viral reverse transcription. HP initiates reverse transcription by using itself as a protein primer (protein priming) and Hε as the obligatory template.

Tobacco-specific nitrosamine-derived O2-alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli.

To investigate the biological effects of the O(2)-alkylthymidines induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), we have replicated a plasmid containing O(2)-methylthymidine (O(2)-Me-dT) or O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine (O(2)-POB-dT) in Escherichia coli with specific DNA polymerase knockouts. High genotoxicity of the adducts was manifested in the low yield of transformants from the constructs, which was 2-5% in most strains but increased 2-4-fold with SOS. In the SOS-induced wild type E.

Characterization of dibenzo[a,l]pyrene-trans-11,12-diol (dibenzo[def,p]chrysene) glucuronidation by UDP-glucuronosyltransferases.

Dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) that has been identified in tobacco smoke and is found in our environment due to incomplete combustion of organic matter. Its metabolites are known to form stable DNA adducts in bacteria and mammalian cells, and can lead to tumors in animal models. Glucuronidation of major metabolites of DB[a,l]P by the uridine-5'-diphosphate glucuronosyltransferase (UGT) family of enzymes is an important route of detoxification of this pro-carcinogen.

Development of novel naphthalimide derivatives and their evaluation as potential melanoma therapeutics.

Synthesis and anti-melanoma activity of various naphthalimide analogs, rationally modified by introducing isothiocyanate (ITC) and thiourea (TU) functionalities, found in well-known anti-cancer agents, is described. The structure-activity relationship comparison of the novel agents in inhibiting cancer cell growth was evaluated in various melanoma cell lines. Both ITC and TU analogs effectively inhibited cell viability and induced apoptosis in various human melanoma cells.

Facile syntheses of O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine, the major adduct formed by tobacco specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in vivo, and its site-specifically adducted oligodeoxynucleotides.

O(2)-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) is the most persistent adduct detected in the lung and liver of rats treated with tobacco specific nitrosamines: N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). It is an important biomarker to assess the human exposure to these carcinogens. The only synthetic method reported for O(2)-POB-dThd requires repeated HPLC purifications and could only be used to prepare an analytical standard due to very low yield (0.

An unprecedented nucleic acid capture mechanism for excision of DNA damage.

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides.

Association studies of excision repair cross-complementation group 1 (ERCC1) haplotypes with lung and head and neck cancer risk in a Caucasian population.

Background: The formation of bulky DNA adducts caused by diol epoxide derivatives of polycyclic aromatic hydrocarbons has been associated with tobacco-induced cancers, and inefficient repair of such adducts by the nucleotide excision repair (NER) system has been linked to increased risk of tobacco-induced lung and head and neck (H&N) cancers. The human excision repair cross-complementation group 1 (ERCC1) protein is essential for a functional NER system and genetic variation in ERCC1 may contribute to impaired DNA repair capacity and increased lung and H&N cancer risk.

Methods: In order to comprehensively capture common genetic variation in the ERCC1 gene, Caucasian data from the International HapMap project was used to assess linkage disequilibrium and choose four tagSNPs (rs1319052, rs3212955, rs3212948, and rs735482) in the ERCC1 gene to genotype 452 lung cancer cases, 175 H&N cancer cases, and 790 healthy controls.

Differential repair of polycyclic aromatic hydrocarbon DNA adducts from an actively transcribed gene.

Polycyclic aromatic hydrocarbons (PAHs) are carcinogens with varying potencies. These compounds are metabolized to diol epoxides that react to form DNA adducts. Nucleotide excision repair is a critical cellular defense against these bulky DNA adducts which, if not repaired, can lead to mutations and the initiation of cancer.

Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase beta and ligase III/XRCC1.

1-Beta-D-arabinofuranosylcytosine (cytarabine, araC) and 2',2'-difluoro-2'-deoxycytidine (gemcitabine, dFdC), are effective cancer chemotherapeutic agents due to their ability to become incorporated into DNA and then subsequently inhibit DNA synthesis by replicative DNA polymerases. However, the impact of these 3'-modified nucleotides on the activity of specialized DNA polymerases has not been investigated. The role of polymerase beta and base excision repair may be of particular importance due to the increased oxidative stress in tumors, increased oxidative stress caused by chemotherapy treatment, and the variable amounts of polymerase beta in tumors.

Functional characterization of low-prevalence missense polymorphisms in the UDP-glucuronosyltransferase 1A9 gene.

The UDP-glucuronosyltransferase (UGT) 1A9 has been shown to play an important role in the detoxification of several carcinogens and clearance of anticancer and pain medications. The goal of the present study was to identify novel polymorphisms in UGT1A9 and characterize their effect on glucuronidation activity. The UGT1A9 gene was analyzed by direct sequencing of buccal cell genomic DNA from 90 healthy subjects.

Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus.

We used a series of dATP and dGTP analogues to determine how DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase, uses N-1, N(2), N-3, and N(6) of purine dNTPs to differentiate between right and wrong nucleotide incorporation. Altering any of these nitrogens had two effects. First, it decreased the efficiency of correct incorporation of the resulting dNTP analogue, with the loss of N-1 and N-3 having the most severe effects.

Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate.

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect.

Identifying the features of purine dNTPs that allow accurate and efficient DNA replication by herpes simplex virus I DNA polymerase.

To accurately replicate its viral genome, the Herpes Simplex Virus 1 (HSV-1) DNA polymerase usually polymerizes the correct natural 2'-deoxy-5'-triphosphate (dNTP) opposite the template base being replicated. We employed a series of purine-dNTP analogues to determine the chemical features of the base necessary for the herpes polymerase to avoid polymerizing incorrect dNTPs. The enzyme uses N-3 to prevent misincorporation of purine dNTPs but does not require N-3 for correct polymerization.

Formation of purine-purine mispairs by Sulfolobus solfataricus DNA polymerase IV.

DNA damage that stalls replicative polymerases can be bypassed with the Y-family polymerases. These polymerases have more open active sites that can accommodate modified nucleotides. The lack of protein-DNA interactions that select for Watson-Crick base pairs correlate with the lowered fidelity of replication.