Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.) via its major adduct [+ta]-B[a]P-N2-dG. We recently showed that the dominant G-to-T mutation depends on DNA polymerase V (DNAP V), but not DNAPs IV or II, when studied in a 5'-TG sequence in E. coli. Herein we investigate what DNAPs are responsible for non-mutagenic bypass with [+ta]-B[a]P-N2-dG, along with its mirror image adduct [-ta]-B[a]P-N2-dG. Each adduct is built into a 5'-TG sequence in a single stranded M13 phage vector, which is then transformed into eight different E. coli strains containing all combinations of proficiency and deficiency in the three lesion-bypass DNAPs II, IV and V. Based on M13 progeny output, non-mutagenic bypass with [-ta]-B[a]P-N2-dG depends on DNAP IV. In contrast, non-mutagenic bypass with [+ta]-B[a]P-N2-dG depends on both DNAPs IV and V, where arguments suggest that DNAP IV is involved in dCTP insertion, while DNAP V is involved in extension of the adduct-G:C base pair. Numerous findings indicate that DNAP II has a slight inhibitory effect on the bypass of [+ta]- and [-ta]-B[a]P-N2-dG in the case of both DNAPs IV and V. In conclusion, for efficient non-mutagenic bypass (dCTP insertion) in E. coli, [+ta]-B[a]P-N2-dG requires DNAPs IV and V, [-ta]-B[a]P-N2-dG requires only DNAP IV, while DNAP II is inhibitory to both, and experiments to investigate these differences should provide insights into the mechanism and purpose of these lesion-bypass DNAPs.
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PMID:Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli. 1648 53

XRCC1 coordinates the activities of DNA polymerase-beta and DNA ligase for base excision repair of oxidative DNA damage. In addition, there is some evidence that XRCC1 is a negative regulator of apoptosis. Single nucleotide polymorphisms in XRCC1 have been inconsistently associated with breast cancer risk. We evaluated XRCC1 gene expression in breast cancer cell lines and carcinogen-induced apoptosis in benign breast epithelial cells in relation to XRCC1 genotypes. XRCC1 IVS10+141G>A was associated with increased expression of XRCC1 mRNA and protein, and reduced apoptosis in response to benzo-[a]-pyrene or ionizing radiation, but XRCC1 R399Q was not. These genotypes were also assessed in a clinic-based sample that included 190 breast cancer patients with a family history of breast cancer and 95 controls with no family history of breast cancer. Heterozygous XRCC1 IVS10+141G>A was associated with an increased breast cancer risk (O.R. = 1.7, 95% C.I. 1.016-2.827, P = 0.04) as was homozygous XRCC1 IVS10+141G>A (O.R. = 4.7, 95% C.I. 1.028-21.444, P = 0.03). XRCC1 R399Q was not associated with breast cancer (O.R. 1.00, 95% C.I. 0.61-1.64). The XRCC1 IVS10+141G>A locus is centered in a sequence that is nearly identical to the consensus binding site for the PLAG1 transcription factor. XRCC1 IVS10+141G>A is an intronic polymorphism that is associated with XRCC1 expression, apoptosis and familial breast cancer. It may occur within an intronic regulatory sequence.
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PMID:An intronic polymorphism associated with increased XRCC1 expression, reduced apoptosis and familial breast cancer. 1659 26

We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polkappa to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polkappa in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polkappa. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polkappa. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polkappa in a DNA damage-independent manner. Therefore, association of Polkappa with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18(-/-) transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18(-/-) cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polkappa. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polkappa to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.
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PMID:Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint-mediated arrest. 1661 94

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. Two orientations of B[a]P-N2-dG (BPmi5 and BPmi3) are considered, along with two orientations of the dCTP (AS1 and AS2), as outlined next. Based on NMR studies, the pyrene moiety of B[a]P-N2-dG is in the minor groove, when paired with dC, and can point toward either the base on the 5'-side (BPmi5) or the 3'-side (BPmi3). Based on published X-ray structures, Dpo4 appears to have two partially overlapping active sites. The architecture of active site 1 (AS1) is similar to all other families of DNAPs (e.g., the shape of the dNTP). Active site 2 (AS2), however, is non-canonical (e.g., the beta- and gamma-phosphates in AS2 are approximately where the alpha- and beta-phosphates are in AS1). In the Dpo4 models generated herein, using the BPmi3 orientation the pyrene moiety of [+ta]-B[a]P-N2-dG points toward the duplex region of the DNA, and is accommodated without distortions in AS1, but with distortions in AS2. Considering the BPmi5 orientation, the pyrene moiety points toward the ss-region of DNA in Dpo4, and sits in a hole defined by the fingers and little fingers domain ("chimney"); BPmi5 is accommodated in AS2 without significant distortions, but poorly in AS1. In summary, when dCTP is paired with [+ta]-B[a]P-N2-dG in the two overlapping active sites in Dpo4, the pyrene in the BPmi3 orientation is accommodated better in active site 1 (AS1), while the pyrene in the BPmi5 orientation is accommodated better in AS2. Finally, we discuss why Y-family DNAPs might have two catalytic active sites.
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PMID:Molecular modeling benzo[a]pyrene N2-dG adducts in the two overlapping active sites of the Y-family DNA polymerase Dpo4. 1678 74

Our previous studies have shown that treatment with cigarette smoke condensate (CSC) transforms normal breast epithelial cell line, MCF-10A. In the present study, the mechanism of CSC-induced transformation of breast epithelial cells was examined. We first determined whether benzo[a]pyrene (B[a]P)- and CSC-induced levels of APC are capable of inhibiting long-patch base excision repair (LP-BER) since our earlier studies had shown that an interaction of APC with DNA polymerase beta (pol-beta) blocks strand-displacement synthesis. With the use of a novel in vivo LP-BER assay, it was demonstrated that increased and decreased APC levels in different breast cancer cell lines were associated with a decrease or increase in LP-BER activity, respectively. The effect of APC on LP-BER in malignant and pre-malignant breast epithelial cell lines was produced by either overexpression or knockdown of APC. Furthermore, it was shown that the decreased LP-BER in B[a]P- or CSC-treated pre-malignant breast epithelial cells is associated with an increased level of APC and decreased cell growth. Our results suggest that the decreased growth allows cells to repair the damaged DNA before mitosis, and failure to repair damaged DNA has the potential to transform pre-malignant breast epithelial cells.
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PMID:Cigarette smoke condensate-induced level of adenomatous polyposis coli blocks long-patch base excision repair in breast epithelial cells. 1692 28

The Y-family DNA polymerase Dpo4, from the thermophilic crenarchaeon Sulfolobus solfataricus P2, offers a valuable opportunity to investigate the effect of conformational flexibility on the bypass of bulky lesions because of its ability to function efficiently at a wide range of temperatures. Combined molecular modeling and experimental kinetic studies have been carried out for 10S-(+)-trans-anti-[BP]-N2-dG ((+)-ta-[BP]G), a lesion derived from the covalent reaction of a benzo[a]pyrene metabolite with guanine in DNA, at 55 degrees C and results compared with an earlier study at 37 degrees C (Perlow-Poehnelt, R. A., Likhterov, I., Scicchitano, D. A., Geacintov, N. E., and Broyde, S. (2004) J. Biol. Chem. 279, 36951-36961). The experimental results show that there is more overall nucleotide insertion opposite (+)-ta-[BP]G due to particularly enhanced mismatch incorporation at 55 degrees C compared with 37 degrees C. The molecular dynamics simulations suggest that mismatched nucleotide insertion opposite (+)-ta-[BP]G is increased at 55 degrees C compared with 37 degrees C because the higher temperature shifts the preference of the damaged base from the anti to the syn conformation, with the carcinogen on the more open major groove side. The mismatched dNTP structures are less distorted when the damaged base is syn than when it is anti, at the higher temperature. However, with the normal partner dCTP, the anti conformation with close to Watson-Crick alignment remains more favorable. The molecular dynamics simulations are consistent with the kcat values for nucleotide incorporation opposite the lesion studied, providing structural interpretation of the experimental observations. The observed temperature effect suggests that conformational flexibility plays a role in nucleotide incorporation and bypass fidelity opposite (+)-ta-[BP]G by Dpo4.
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PMID:Increased flexibility enhances misincorporation: temperature effects on nucleotide incorporation opposite a bulky carcinogen-DNA adduct by a Y-family DNA polymerase. 1709 May 33

Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic Hus1 allele, Hus1(neo), with either wild-type or null (Hus1(Delta1)) alleles. Primary Hus1(neo/Delta1) embryonic fibroblasts exhibited spontaneous chromosomal abnormalities and underwent premature senescence, while higher Hus1 expression in Hus1(neo/neo) cells allowed for normal proliferation. Antioxidant treatment almost fully suppressed premature senescence in Hus1(neo/Delta1) cultures, suggesting a critical role for Hus1 in oxidative stress responses. Treatment of Hus1(neo/neo) and Hus1(neo/Delta1) cells with the DNA adducting agent benzo(a)pyrene dihydrodriol epoxide resulted in a loss of cell viability that was associated with S-phase DNA damage checkpoint failure. Likewise, the DNA polymerase inhibitor aphidicolin triggered increased cell death, chromosomal aberrations, and H2AX phosphorylation, a marker for double-stranded DNA breaks, in Hus1(neo/neo) and Hus1(neo/Delta1) cultures compared to controls. Despite these pronounced genome maintenance defects in cultured Hus1(neo/Delta1) and Hus1(neo/neo) cells, mice of the same genotypes were born at expected frequencies and appeared grossly normal. A significant increase in micronucleus formation was observed in peripheral blood cells from Hus1(neo/Delta1) mice, but reduced Hus1 expression did not cause an elevated predisposition to spontaneous tumor development or accelerate tumorigenesis in p53-deficient mice. These results identify differential effects of altered Hus1 gene dosage on genome maintenance during in vitro culture, genotoxic stress responses, embryonic development, and adult homeostasis.
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PMID:Genome maintenance defects in cultured cells and mice following partial inactivation of the essential cell cycle checkpoint gene Hus1. 1722 Feb 76

We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.
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PMID:Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase. 1757 77

The Y-family DNA polymerase Dpo4, from the archaeon bacterium Sulfolobus solfataricus, is a member of the DinB family, which also contains human Pol kappa. It has a spacious active site that can accommodate two templating bases simultaneously, with one of them skipped by the incoming dNTP. Assays of single dNTP insertion opposite a benzo[ a]pyrene-derived N (2)-dG adduct, 10 S(+)- trans- anti-[BP]- N (2)-dG ([BP]G*), reveal that an incoming dATP is significantly preferred over the other three dNTPs in the TG 1*G 2 sequence context. Molecular modeling and dynamics simulations were carried out to interpret this experimental observation on a molecular level. Modeling studies suggest that the significant preference for dATP insertion observed experimentally can result from two possible dATP incorporation modes. The dATP can be inserted opposite the T on the 5' side of the adduct G 1*, using an unusual 5'-slippage pattern, in which the unadducted G 2, rather than G 1*, is skipped, to produce a -1 deletion. In addition, the dATP can be misincorporated opposite the adduct. The 5'-slippage pattern may be generally facilitated in cases where the base 3' to the lesion is the same as the adducted base.
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PMID:Nucleotide selectivity opposite a benzo[a]pyrene-derived N2-dG adduct in a Y-family DNA polymerase: a 5'-slippage mechanism. 1826 Jun 44

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.
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PMID:Lesion processing: high-fidelity versus lesion-bypass DNA polymerases. 1840 2


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