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1.
Cancer Genet ; 260-261: 30-36, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34852986

RESUMO

BACKGROUND: It is likely that additional genes for hereditary breast cancer can be identified using a discordant sib pair design. Using this design we identified individuals harboring a rare PMS1 c.605G>A variant previously predicted to result in loss of function. OBJECTIVES: A family-based design and predictive algorithms were used to prioritize candidate variants possibly associated with an increased risk of hereditary breast cancer. Functional analyses were performed for one of the candidate variants, PMS1 c.605G>A. METHODS: 1) 14 discordant sister-pairs from hereditary breast cancer families were identified. 2) Whole exome sequencing was performed and candidate risk variants identified. 3) A rare PMS variant was identified in 2 unrelated affected sisters but no unaffected siblings. 4) Functional analysis of this variant was carried out using targeted mRNA sequencing. RESULTS: Genotype-phenotype correlation did not demonstrate tracking of the variant with cancer in the family. Functional analysis revealed no difference in exon 6 incorporation, which was validated by analyzing PMS1 allele specific expression. CONCLUSIONS: The PMS1 c.605G>A variant did not segregate with disease, and there was no variant-dependent impact on PMS1 exon 6 splicing, supporting this variant is likely benign. Functional analyses are imperative to understanding the clinical significance of predictive algorithms.


Assuntos
Neoplasias da Mama/genética , Sequenciamento do Exoma/métodos , Perfilação da Expressão Gênica/métodos , Proteínas MutL/genética , Proteínas de Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Adulto , Algoritmos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Mutação com Perda de Função , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de RNA , Irmãos
2.
DNA Repair (Amst) ; 57: 91-97, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28709015

RESUMO

Reactive oxygen species generate some 20,000 base lesions per human cell per day. The vast majority of these potentially mutagenic or cytotoxic lesions are subject to base excision repair (BER). Although chromatin remodelers have been shown to enhance the excision of oxidized bases from nucleosomes in vitro, it is not clear that they are recruited to and act at sites of BER in vivo. To test the hypothesis that cells possess factors that enhance BER in chromatin, we assessed the capacity of nuclear extracts from human cells to excise thymine glycol (Tg) lesions from exogenously added, model nucleosomes. The DNA glycosylase NTHL1 in these extracts was able to excise Tg from both naked DNA and sites in nucleosomes that earlier studies had shown to be sterically accessible. However, the same extracts were able to excise lesions from sterically-occluded sites in nucleosomes only after the addition of Mg2+/ATP. Gel mobility shift assays indicated that nucleosomes remain largely intact following the Mg2+/ATP -dependent excision reaction. Size exclusion chromatography indicated that the NTHL1-stimulating activity has a relatively low molecular weight, close to that of NTHL1 and other BER glycosylases; column fractions that contained the very large chromatin remodeling complexes did not exhibit this same stimulatory activity. These results indicate that cells possess a factor(s) that promotes the initiation of BER in chromatin, but differs from most known chromatin remodeling complexes.


Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Nucleossomos/metabolismo , Timina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Cátions Bivalentes/metabolismo , Linhagem Celular , DNA/metabolismo , Humanos , Magnésio/metabolismo , Timina/metabolismo
3.
Oncogenesis ; 3: e128, 2014 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-25417725

RESUMO

Helicobacter pylori infection of the human stomach is associated with inflammation that leads to the release of reactive oxygen and nitrogen species (RONs), eliciting DNA damage in host cells. Unrepaired DNA damage leads to genomic instability that is associated with cancer. Base excision repair (BER) is critical to maintain genomic stability during RONs-induced DNA damage, but little is known about its role in processing DNA damage associated with H. pylori infection of normal gastric epithelial cells. Here, we show that upon H. pylori infection, abasic (AP) sites accumulate and lead to increased levels of double-stranded DNA breaks (DSBs). In contrast, downregulation of the OGG1 DNA glycosylase decreases the levels of both AP sites and DSBs during H. pylori infection. Processing of AP sites during different phases of the cell cycle leads to an elevation in the levels of DSBs. Therefore, the induction of oxidative DNA damage by H. pylori and subsequent processing by BER in normal gastric epithelial cells has the potential to lead to genomic instability that may have a role in the development of gastric cancer. Our results are consistent with the interpretation that precise coordination of BER processing of DNA damage is critical for the maintenance of genomic stability.

4.
Chromosoma ; 110(6): 402-10, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11734998

RESUMO

DNA polymerase beta (pol beta) is an essential enzyme that has been shown to localize as discrete foci to the synaptonemal complex during meiosis in the mouse. To identify proteins that associate with pol beta during meiosis, we employed the yeast two-hybrid screen. Here we show that a multiple PDZ domain-containing protein, the glutamate receptor interacting protein 1 (GRIP1), interacts specifically with pol beta. The PDZ domain-containing proteins, including GRIP1, act as scaffolds to promote rapid and localized biochemical events that require the interaction of multiple proteins. GRIP1 localizes to discrete foci on meiotic bivalents of both spermatocyte and oocyte nuclei, and colocalizes with pol beta. Together, these findings provide evidence that GRIP1 interacts with pol beta during meiosis. Our findings are consistent with the possibility that GRIP1 acts as a scaffold to promote interaction between proteins that function during meiosis.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Meiose , Fatores de Transcrição/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Modelos Genéticos , Coativador 2 de Receptor Nuclear , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Testículo/metabolismo , Técnicas do Sistema de Duplo-Híbrido
5.
Biochemistry ; 40(38): 11372-81, 2001 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-11560485

RESUMO

DNA polymerase beta (pol beta) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol beta (M282Lbeta) was identified using an in vivo genetic screen. Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on alpha-helix N of pol beta. This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays. M282Lbeta has a 7.5-fold higher mutation frequency than wild-type pol beta; M282Lbeta commits a variety of base substitution and frameshift errors. Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lbeta. Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding. However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved. X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity. Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain. Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282. Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair. The structural changes of M282Lbeta ultimately lead to an overall reduction in polymerase fidelity.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , DNA Bacteriano/química , Desoxirribonucleotídeos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Mutação , Substituição de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , DNA Polimerase beta/genética , DNA Bacteriano/genética , Estabilidade Enzimática , Mutação da Fase de Leitura , Biblioteca Gênica , Genótipo , Temperatura Alta , Cinética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Mutação Puntual , Conformação Proteica , Desnaturação Proteica , Simplexvirus/enzimologia , Simplexvirus/genética , Especificidade por Substrato , Termodinâmica , Timidina Quinase/genética , Ureia
6.
J Biol Chem ; 276(14): 10824-31, 2001 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-11154692

RESUMO

DNA polymerases have the unique ability to select a specific deoxynucleoside triphosphate from a pool of similarly structured substrates. One of these enzymes, DNA polymerase beta, offers a simple system to relate polymerase structure to the fidelity of DNA synthesis. In this study, a mutator DNA polymerase beta, Y265H, was identified using an in vivo genetic screen. Purified Y265H produced errors at a 40-fold higher frequency than the wild-type protein in a forward mutation assay. At 37 degrees C, transient kinetic analysis demonstrated that the alteration caused a 111-fold decrease in the maximum rate of polymerization and a 117-fold loss in fidelity for G misincorporation opposite template A. Our data suggest that the maximum rate of polymerization was reduced, because Y265H was dramatically impaired in its ability to perform nucleotidyl transfer in the presence of the correct nucleotide substrate. In contrast, at 20 degrees C, the mutant protein had a fidelity similar to wild-type enzyme. Both proteins at 20 degrees C demonstrate a rapid change in protein conformation, followed by a slow chemical step. These data suggest that proper geometric alignment of template, 3'-OH of the primer, magnesium ions, dNTP substrates, and the active site residues of DNA polymerase beta are important factors in polymerase fidelity and provide the first evidence that Tyr-265 is important for this alignment to occur properly in DNA polymerase beta.


Assuntos
DNA Polimerase beta/genética , Mutação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Polimerase beta/química , Conformação Proteica , Relação Estrutura-Atividade
7.
J Biol Chem ; 274(50): 35866-72, 1999 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-10585471

RESUMO

The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Substituição de Aminoácidos , Sequência de Bases , DNA/química , DNA/metabolismo , DNA Polimerase beta/genética , Primers do DNA , Ácido Glutâmico , Cinética , Lisina , Modelos Moleculares , Mutagênese , Conformação de Ácido Nucleico , Fenótipo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Zidovudina/farmacocinética , Zidovudina/farmacologia
8.
Proc Natl Acad Sci U S A ; 96(17): 9580-5, 1999 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-10449735

RESUMO

DNA polymerase beta functions in both base excision repair and meiosis. Errors committed by polymerase beta during these processes could result in mutations. Using a complementation system, in which rat DNA polymerase beta substitutes for DNA polymerase I of Escherichia coli, we previously isolated a DNA polymerase beta mutant in which Tyr-265 was altered to Cys (Y265C). The Y265C mutant is dominant to wild-type DNA polymerase beta and possesses an intrinsic mutator activity. We now have expressed the wild-type DNA polymerase and the Y265C mutator mutant in mouse LN12 cells, which have endogenous DNA polymerase beta activity. We demonstrate that expression of the Y265C mutator mutant in the LN12 cells results in an 8-fold increase in the spontaneous mutation frequency of lambdacII mutants compared with expression of the wild-type protein. Expression of Y265C results in at least a 40-fold increase in the frequency of deletions of three bases or more and a 7-fold increase in point mutations. Our results suggest that the mutations we observe in vivo result directly from the action of the mutator polymerase. To our knowledge, this is the first demonstration of a mutator phenotype resulting from expression of a DNA polymerase mutator mutant in mammalian cells. This work raises the possibility that variant polymerases may act in a dominant fashion in human cells, leading to genetic instability and carcinogenesis.


Assuntos
Cisteína , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Mutagênese , Tirosina , Substituição de Aminoácidos , Animais , Sequência de Bases , DNA Polimerase beta/química , Mutação da Fase de Leitura , Humanos , Camundongos , Dados de Sequência Molecular , Fenótipo , Ratos , Deleção de Sequência
9.
Biochemistry ; 38(15): 4800-8, 1999 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-10200168

RESUMO

DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.


Assuntos
DNA Polimerase beta/metabolismo , Desoxirribonucleotídeos/metabolismo , Fenilalanina/metabolismo , Pareamento Incorreto de Bases , Sequência de Bases , DNA Polimerase beta/química , Modelos Moleculares , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
10.
J Biol Chem ; 274(6): 3851-8, 1999 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-9920940

RESUMO

We developed an in vivo selection to identify 3'-azido-3'-deoxythymidine (AZT)-resistant mutants of rat DNA polymerase beta (pol beta). The selection utilizes pol beta's ability to substitute for Escherichia coli DNA polymerase I (pol I) in the SC18-12 strain, which lacks active pol I. pol beta allows SC18-12 cells to grow, but they depend on pol beta activity, so inhibition of pol beta by AZT kills them. We screened a library of randomly mutated pol beta cDNA for complementation of the pol I defect in the presence of AZT, and identified AZT-resistant mutants. We purified two enzymes with nonconservative mutations in the palm domain of the polymerase. The substitutions D246V and R253M result in reductions in the steady-state catalytic efficiency (Kcat/Km) of AZT-TP incorporation. The efficiency of dTTP incorporation was unchanged for the D246V enzyme, indicating that the substantial decrease in AZT-TP incorporation is responsible for its drug resistance. The R253M enzyme exhibits significantly higher Km(dTTP) and Kcat(dTTP) values, implying that the incorporation reaction is altered. These are the first pol beta mutants demonstrated to exhibit AZT resistance in vitro. The locations of the Asp-246 and Arg-253 side chains indicate that substrate specificity is influenced by residues distant from the nucleotide-binding pocket.


Assuntos
DNA Polimerase beta/genética , Mutação , Zidovudina/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Sequência de Bases , Catálise , Linhagem Celular , DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Primers do DNA , Resistência Microbiana a Medicamentos , Teste de Complementação Genética , Fenótipo , Conformação Proteica , Ratos , Especificidade por Substrato , Zidovudina/metabolismo
11.
J Bacteriol ; 180(9): 2292-7, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9573177

RESUMO

We demonstrated recently that dominant negative mutants of rat DNA polymerase beta (Pol beta) interfere with repair of alkylation damage in Saccharomyces cerevisiae. To identify the alkylation repair pathway that is disrupted by the Pol beta dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol beta mutants to genes known to be involved in repair of DNA alkylation damage in S. cerevisiae. We demonstrate that the rat Pol beta mutants interfere with the base excision repair pathway in S. cerevisiae. In addition, expression of one of the Pol beta dominant negative mutants, Pol beta-14, increases the spontaneous mutation rate of S. cerevisiae whereas expression of another Pol beta dominant negative mutant, Pol beta-TR, does not. Expression of the Pol beta-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate. These results suggest that gaps are required for mutagenesis to occur in the presence of Pol beta-14 but that it is not merely the presence of a gap that results in mutagenesis. Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.


Assuntos
DNA Glicosilases , DNA Polimerase beta/genética , Reparo do DNA , Mutagênese , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Alquilação , Animais , Dano ao DNA , DNA Polimerase beta/metabolismo , Enzimas Reparadoras do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Epistasia Genética , Resolvases de Junção Holliday , Mutação , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Ratos
12.
Biochemistry ; 37(8): 2111-9, 1998 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-9485358

RESUMO

This study describes the first complete in vitro error specificity analysis of a mutator DNA polymerase that is altered in a residue not predicted to contact either the DNA or dNTP substrate. We examined this mutator form of polymerase beta (Y265C) in order to elucidate the critical role tyrosine 265 plays in the accuracy of DNA synthesis. Our results demonstrate that an increase in both frame shift errors in homonucleotide repeat sequences and base substitution errors contribute nearly equally to the Y265C mutator phenotype. The models described for production of these errors, primer/template misalignment and base misincorporation, respectively, are distinctly different, suggesting the Y265C alteration affects discrimination against both types of error production pathways. In addition, Y265C displays a 530-fold increase in multiple errors within the 203-base pair target region examined, relative to that of wild type. Processivity studies revealed that Y265C retains the near distributive nature of DNA synthesis characteristic of the wild type polymerase beta. Therefore, multiple errors exhibited by Y265C most likely result from independent polymerase binding events. Localization of tyrosine 265 in the X-ray crystallographic structure suggests this residue may play a role in mediating a conformational change of the polymerase [Pelletier, H., et al. (1996) Biochemistry 35, 12742-12761]. A conformational change is predicted to enhance the accuracy of DNA synthesis by imposing an induced fit selection against premutational intermediates. The observed loss of discrimination against both misalignment-mediated and misincorporation-mediated errors produced by polymerase Y265C is consistent with such a model.


Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , DNA/biossíntese , DNA/genética , DNA Polimerase beta/metabolismo , Reparo do DNA , Mutação da Fase de Leitura , Óperon Lac , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , Ratos , Simplexvirus/genética , Timidina Quinase/genética , Tirosina/química
13.
J Biol Chem ; 272(44): 27501-4, 1997 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-9346877

RESUMO

DNA polymerase beta (pol beta) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol beta activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol beta (kcat/Km,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5'-margin of the gap. pol beta exhibited the highest catalytic efficiency on 5'-phosphorylated 1-nucleotide gapped DNA. This efficiency was >/=500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol beta, as judged by its ability to form G-N mispairs, was also higher (10-100 times) on 5'-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol beta is to fill 5'-phosphorylated 1-nucleotide gaps.


Assuntos
DNA Polimerase beta/metabolismo , DNA/metabolismo , Animais , Catálise , Cinética , Dados de Sequência Molecular , Fosforilação , Ratos , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
14.
Proc Natl Acad Sci U S A ; 94(4): 1321-6, 1997 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-9037051

RESUMO

DNA polymerase beta (pol beta) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol beta mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol beta to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol beta mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol beta to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol beta-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol beta-14 and another of our mutant proteins, pol beta-166, is probably critical for accurate DNA synthesis by pol beta. Thus, our genetic method of screening for pol beta mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol beta enzyme.


Assuntos
DNA Polimerase I/genética , Replicação do DNA/genética , Teste de Complementação Genética , Mutagênese/genética , Seleção Genética , Animais , Escherichia coli/genética , Fenótipo , Ratos , Proteínas Recombinantes , Tirosina/genética
15.
Proc Natl Acad Sci U S A ; 94(4): 1327-31, 1997 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-9037052

RESUMO

DNA polymerase beta (pol beta) is an enzyme possessing both polymerase and deoxyribose phophatase activities. Although pol beta is not believed to participate in the replication of genomic DNA, several studies have indicated a role for pol beta in DNA repair. The high level of expression of pol beta in mouse and rat testes raises the possibility that pol beta participates in mammalian meiosis. Using antibody localization, we detect foci that stain with pol beta antisera at discrete sites along homologous chromosomes as they synapse and progress through prophase of meiosis I. These data suggest that pol beta participates in meiotic events associated with synapsis and recombination.


Assuntos
Cromossomos/enzimologia , DNA Polimerase I/isolamento & purificação , Complexo Sinaptonêmico , Testículo/enzimologia , Animais , Diferenciação Celular , DNA Polimerase I/imunologia , Imuno-Histoquímica , Masculino , Camundongos , Recombinação Genética , Espermatócitos/enzimologia
16.
J Bacteriol ; 178(3): 656-61, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8550496

RESUMO

DNA polymerase beta is one of the smallest known eukaryotic DNA polymerases. This polymerase has been very well characterized in vitro, but its functional role in vivo has yet to be determined. Using a novel competition assay in Escherichia coli, we isolated two DNA polymerase beta dominant negative mutants. When we overexpressed the dominant negative mutant proteins in Saccharomyces cerevisiae, the cells became sensitive to methyl methanesulfonate. Interestingly, overexpression of the same polymerase beta mutant proteins did not confer sensitivity to UV damage, strongly suggesting that the mutant proteins interfere with the process of base excision repair but not nucleotide excision repair in S. cerevisiae. Our data implicate a role for polymerase IV, the S. cerevisiae polymerase beta homolog, in base excision repair in S. cerevisiae.


Assuntos
DNA Polimerase I/fisiologia , Reparo do DNA , Saccharomyces cerevisiae/genética , Animais , Escherichia coli/genética , Metanossulfonato de Metila/farmacologia , Mutação , Ratos , Raios Ultravioleta
17.
Mol Gen Genet ; 248(2): 217-24, 1995 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-7651344

RESUMO

We used quantitative complementation assays to characterize individual DNA polymerase beta (Pol beta) mutants for their ability to function in DNA replication and DNA repair. We also describe a screen for detecting mutator activity of DNA polymerase beta mutants. By using these bioassays, together with DNA polymerase activity gels, we characterized 15 new DNA polymerase beta mutants that display a wide spectrum of phenotypes. Most of these mutants are generally defective in their ability to synthesize DNA. However, two of our Pol beta mutants show more complex phenotypes: they are able to function in DNA repair but unable to participate in DNA replication. One of our mutants displays mutator activity in vivo. Our work provides a model to study mutant mammalian enzymes in Escherichia coli with phenotypes that are otherwise difficult to assess.


Assuntos
DNA Polimerase I/genética , Animais , Western Blotting , DNA Polimerase I/química , DNA Polimerase I/metabolismo , Reparo do DNA , Replicação do DNA , Escherichia coli/genética , Teste de Complementação Genética , Metanossulfonato de Metila/farmacologia , Mutagênese , Mutação , Ácido Nitroso/farmacologia , Fenótipo , Ratos
18.
J Bacteriol ; 177(10): 2923-5, 1995 May.
Artigo em Inglês | MEDLINE | ID: mdl-7751308

RESUMO

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.


Assuntos
DNA Polimerase I/metabolismo , Replicação do DNA , Escherichia coli/genética , Plasmídeos/biossíntese , Resistência a Ampicilina/genética , Animais , Sequência de Bases , DNA Polimerase I/genética , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Especificidade da Espécie
19.
Hum Genet ; 93(5): 507-12, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-8168825

RESUMO

Werner syndrome (WS) is a rare autosomal recessive disorder of humans characterized by the premature onset and accelerated rate of development of several major age-related disorders. An aberration in DNA replication or repair is suggested by the evidence of genome instability. Since the structural gene for DNA polymerase beta maps within the region of the WS mutation on the short arm of chromosome 8 and is involved in both DNA repair and DNA replication, we evaluated its candidacy as the WS gene. Several independent lines of evidence did not support that hypothesis: (1) activity gels showed normal enzyme activity and electrophoretic mobility; (2) nucleotide sequence analysis of the entire coding region failed to reveal mutations (although indicated mistakes in the published sequence); (3) single-strand conformation polymorphism (SSCP) and heteroduplex analyses failed to reveal evidence of mutations in the promoter region; (4) a newly discerned polymorphism failed to reveal evidence of homozygosity by descent in a consanguineous patient; and 5) fluorescence in situ hybridization (FISH) analysis placed the DNA polymerase beta gene centromeric to D8S135 at 8p11.2 and thus beyond the region of peak LOD scores for WS.


Assuntos
DNA Polimerase I/genética , Síndrome de Werner/genética , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 8 , Consanguinidade , DNA Polimerase I/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Hibridização in Situ Fluorescente , Masculino , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Genético , Sequências Reguladoras de Ácido Nucleico , Síndrome de Werner/enzimologia
20.
Proc Natl Acad Sci U S A ; 90(10): 4626-30, 1993 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-8506308

RESUMO

We have designed and utilized a bacterial complementation system to identify and characterize mammalian DNA polymerase beta mutants. In this complementation system, wild-type rat DNA polymerase beta replaces both the replicative and repair functions of DNA polymerase I in the Escherichia coli recA718 polA12 double mutant; our 263 DNA polymerase beta mutants replace E. coli polymerase I less efficiently or not at all. Of the 10 mutants that have been shown to contain DNA sequence alterations, 2 exhibit a split phenotype with respect to complementation of the growth defect and methylmethanesulfonate sensitivity of the double mutant; one is a null mutant. The mutants possessing a split phenotype contain amino acid residue alterations within a putative nucleotide binding site of DNA polymerase beta. This approach for the isolation and evaluation of mutants of a mammalian DNA polymerase in E. coli may ultimately lead to a better understanding of the mechanism of action of this enzyme and to precisely defining its role in vertebrate cells.


Assuntos
DNA Polimerase I/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Reparo do DNA , Replicação do DNA , Escherichia coli/genética , Genes , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos/química , Ratos
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