RESUMO
High affinity antibodies are generated in mice and humans by means of somatic hypermutation (SHM) of variable (V) regions of Ig genes. Mutations with rates of 10(-5)--10(-3) per base pair per generation, about 10(6)-fold above normal, are targeted primarily at V-region hot spots by unknown mechanisms. We have measured mRNA expression of DNA polymerases iota, eta, and zeta by using cultured Burkitt's lymphoma (BL)2 cells. These cells exhibit 5-10-fold increases in heavy-chain V-region mutations targeted only predominantly to RGYW (R = A or G, Y = C or T, W = T or A) hot spots if costimulated with T cells and IgM crosslinking, the presumed in vivo requirements for SHM. An approximately 4-fold increase pol iota mRNA occurs within 12 h when cocultured with T cells and surface IgM crosslinking. Induction of pols eta and zeta occur with T cells, IgM crosslinking, or both stimuli. The fidelity of pol iota was measured at RGYW hot- and non-hot-spot sequences situated at nicks, gaps, and double-strand breaks. Pol iota formed T x G mispairs at a frequency of 10(-2), consistent with SHM-generated C to T transitions, with a 3-fold increased error rate in hot- vs. non-hot-spot sequences for the single-nucleotide overhang. The T cell and IgM crosslinking-dependent induction of pol iota at 12 h may indicate an SHM "triggering" event has occurred. However, pols iota, eta, and zeta are present under all conditions, suggesting that their presence is not sufficient to generate mutations because both T cell and IgM stimuli are required for SHM induction.
Assuntos
Linfócitos B/imunologia , DNA Polimerase Dirigida por DNA/genética , Região Variável de Imunoglobulina/genética , Mutação , Animais , Linfócitos B/patologia , Genes de Imunoglobulinas , Humanos , Camundongos , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
The UmuD'2C protein complex (Escherichia coli pol V) is a low-fidelity DNA polymerase (pol) that copies damaged DNA in the presence of RecA, single-stranded-DNA binding protein (SSB) and the beta,gamma-processivity complex of E. coli pol III (ref. 4). Here we propose a model to explain SOS-lesion-targeted mutagenesis, assigning specific biochemical functions for each protein during translesion synthesis. (SOS lesion-targeted mutagenesis occurs when pol V is induced as part of the SOS response to DNA damage and incorrectly incorporates nucleotides opposite template lesions.) Pol V plus SSB catalyses RecA filament disassembly in the 3' to 5' direction on the template, ahead of the polymerase, in a reaction that does not involve ATP hydrolysis. Concurrent ATP-hydrolysis-driven filament disassembly in the 5' to 3' direction results in a bidirectional stripping of RecA from the template strand. The bidirectional collapse of the RecA filament restricts DNA synthesis by pol V to template sites that are proximal to the lesion, thereby minimizing the occurrence of untargeted mutations at undamaged template sites.
Assuntos
Trifosfato de Adenosina/análogos & derivados , DNA Bacteriano/genética , Escherichia coli/genética , Mutagênese , Resposta SOS em Genética , Trifosfato de Adenosina/metabolismo , Catálise , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonuclease I/metabolismo , Proteínas de Escherichia coli , Modelos Genéticos , Recombinases Rec A/metabolismoRESUMO
Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta. gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA ( approximately 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only approximately 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA Polimerase III/química , DNA/química , Escherichia coli/metabolismo , Holoenzimas/química , HidróliseRESUMO
The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol delta alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs. The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged. The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol delta HE with E. coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.
Assuntos
DNA Polimerase III/química , DNA Polimerase III/metabolismo , Exonucleases/metabolismo , Proteínas de Homeodomínio , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/enzimologia , Sequência de Bases , Primers do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C , Schizosaccharomyces/genética , Moldes GenéticosRESUMO
The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.
Assuntos
DNA Polimerase III/metabolismo , Escherichia coli/enzimologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Ligação Competitiva , Primers do DNA , Cinética , Especificidade por Substrato , Moldes GenéticosRESUMO
Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA/genética , DNA/metabolismo , Animais , Dano ao DNA , DNA Polimerase Dirigida por DNA/classificação , Humanos , Mutação , Neoplasias Cutâneas/genética , Moldes GenéticosRESUMO
The beta sliding clamp encircles DNA and enables processive replication of the Escherichia coli genome by DNA polymerase III holoenzyme. The clamp loader, gamma complex, assembles beta around DNA in an ATP-fueled reaction. Previous studies have shown that gamma complex opens the beta ring and also interacts with DNA on binding ATP. Here, a rapid kinetic analysis demonstrates that gamma complex hydrolyzes two ATP molecules sequentially when placing beta around DNA. The first ATP is hydrolyzed fast, at 25-30 s(-1), while the second ATP hydrolysis is limited to the steady-state rate of 2 s(-1). This step-wise reaction depends on both primed DNA and beta. DNA alone promotes rapid hydrolysis of two ATP molecules, while beta alone permits hydrolysis of only one ATP. These results suggest that beta inserts a slow step between the two ATP hydrolysis events in clamp assembly, during which the clamp loader may perform work on the clamp. Moreover, one ATP hydrolysis is sufficient for release of beta from the gamma complex. This implies that DNA-dependent hydrolysis of the other ATP is coupled to a separate function, perhaps involving work on DNA. A model is presented in which sequential ATP hydrolysis drives distinct events in the clamp-assembly pathway. We also discuss underlying principles of this step-wise mechanism that may apply to the workings of other ATP-fueled biological machines.
Assuntos
Trifosfato de Adenosina/metabolismo , DNA Polimerase III/metabolismo , DNA Bacteriano/metabolismo , Sequência de Bases , Primers do DNA/genética , Replicação do DNA , DNA Bacteriano/genética , Metabolismo Energético , Escherichia coli/genética , Escherichia coli/metabolismo , Holoenzimas/metabolismo , Hidrólise , Cinética , Modelos Biológicos , Proteínas Motores MolecularesRESUMO
The beta protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the gamma complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA. Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), have been used to show that ATP binding is required for gamma complex and beta to associate with DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for gamma complex to release the beta.DNA complex and complete the reaction. In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta. However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS in reactions using wild type beta. Thus, the assembly of beta around DNA is coupled tightly to the ATPase activity of the gamma complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.
Assuntos
DNA Polimerase III/química , DNA Polimerase III/metabolismo , DNA Bacteriano/metabolismo , DNA/química , DNA/metabolismo , Escherichia coli/enzimologia , Conformação de Ácido Nucleico , Conformação Proteica , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Substituição de Aminoácidos , Sequência de Bases , DNA Polimerase III/genética , DNA Bacteriano/química , Polarização de Fluorescência , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
DNA synthesis and postreplication mismatch repair were measured in vitro using cell-free extracts from cultured human SY5Y neuroblastoma and WI38 fibroblast cells in different growth states. All extracts, including differentiated SY5Y and quiescent WI38 fibroblasts, catalyzed SV40 origin-dependent DNA synthesis, totally dependent on SV40 T-antigen. Thus, although differentiated neuroblastoma and quiescent fibroblasts cells were essentially nondividing, their extracts were competent for DNA replication using DNA polymerases delta, alpha, and possibly epsilon, with proliferating cell nuclear antigen. Nonreplicative DNA synthesis and lesion bypass by either alpha- or beta-polymerases were detected independently in extracts using primed or gapped single-stranded DNA templates. Long-patch postreplication mismatch repair was measured for the first time in neuroblastoma cell-free extracts. Extracts from subconfluent and high-density SY5Y cells catalyzed postreplication mismatch repair with efficiencies comparable to those of HeLa cell extracts. No significant differences were observed in repair between SY5Y differentiated and undifferentiated cell extracts. Mismatch repair efficiencies were threefold lower in extracts from subconfluent WI38 cells, and repair in WI38 quiescent cells was fourfold less than in subconfluent cells, suggesting that mismatch repair may be regulated. The spectrum of mismatch repair in SY5Y extracts closely resembled the mismatch removal specificities of HeLa extracts: T . G and G . G mismatches were repaired most efficiently; C . A, A . A, A . G and a five-base loop were repaired with intermediate efficiency; repair of G . A, C . C, and T . T mismatches was extremely inefficient.
Assuntos
Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Neuroblastoma , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Extratos Celulares/genética , DNA Ligases/fisiologia , DNA Polimerase I/metabolismo , DNA Polimerase beta/metabolismo , DNA Viral/genética , Fibroblastos/citologia , Fibroblastos/enzimologia , Células HeLa , Humanos , Origem de Replicação/genética , Vírus 40 dos Símios/genética , Tretinoína/farmacologiaRESUMO
A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.
Assuntos
DNA Polimerase III/metabolismo , Proteínas de Ligação a DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Bases , DNA Polimerase III/química , Replicação do DNA , Proteínas de Ligação a DNA/química , Escherichia coli/enzimologia , Polarização de Fluorescência , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Ligação Proteica , Moldes GenéticosRESUMO
Gemcitabine [2',2'-difluorodeoxycytidine (dFdCyd)], a potent antitumor agent, inhibits DNA synthesis and is incorporated internally into DNA. The effect of a template-incorporated dFdCyd molecule (dFdCyd-) on DNA polymerase function was examined. Two 25-base deoxyoligonucleotides were synthesized with either a single dFdCyd- or template-incorporated deoxycytidine molecule (dCyd-) at the same position. Each was annealed separately to an identical complementary 5'-32P-labeled primer and extended by the Klenow fragment (3'-->5' exo-) of DNA polymerase I. "Correct" insertion of dGMP was 80-fold less efficient opposite dFdCyd- than dCyd-. A comparison of misinsertion efficiencies opposite template dFdCyd gave values of 2.7 x 10(-2) for dAMP insertion, 1.1 x 10(-3) for dTMP insertion, and 5.9 x 10(-4) for dCMP insertion. A similar measurement opposite template dC gave values of 1.8 x 10(-4), 1.7 x 10(-4), and 2.9 x 10(-6) for dAMP, dTMP, and dCMP insertion, respectively. Thus, the presence of dFdCyd on the template strand inhibited "normal" DNA synthesis and increased deoxyribonucleotide misinsertion frequencies. Pausing during DNA synthesis occurred directly opposite template dFdCyd suggesting that dFdC.dG base pairs might be less stable than normal dC.dG pairs, resulting in a decreased rate of primer extension beyond this site. Consistent with kinetic data, thermal denaturation measurements using comparable surrounding sequences showed that dFdC.dG "correct" pairs were less stable than dC.dG base pairs. Measurements on base mispairs showed that dFdC.dC was more stable than dC.dC, while no measurable Tm differences were found between polymers containing dFdC.dA and dC.dA or dFdC.dT, and dC.dT.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , DNA Polimerase I/metabolismo , Desoxicitidina/análogos & derivados , Composição de Bases , Sequência de Bases , Clonagem Molecular , DNA Polimerase I/genética , Primers do DNA , Desoxicitidina/farmacologia , Desoxirribonucleotídeos/metabolismo , Exodesoxirribonuclease V , Exodesoxirribonucleases/genética , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Moldes Genéticos , GencitabinaRESUMO
We measured the insertion fidelity of DNA polymerases alpha and beta and yeast DNA polymerase I at a template site that was previously observed to yield a high frequency of T----G transversions when copied by DNA polymerase beta but not by the other two polymerases. The results provide direct biochemical evidence that base substitution errors by DNA polymerase beta can result from a dislocation mechanism governed by DNA template-primer misalignment. In contrast to DNA polymerase beta, neither Drosophila DNA polymerase alpha nor yeast DNA polymerase I appear to misinsert nucleotides by a dislocation mechanism in either the genetic or kinetic fidelity assays. Dislocation errors by DNA polymerase beta are characterized primarily by a substantial reduction in the apparent Km for inserting a "correct," but ultimately errant, nucleotide compared to the apparent Km governing direct misinsertion. For synthesis by DNA polymerase beta, dislocation results in a 35-fold increase in dCMP incorporation opposite template T (T----G transversion) and a 20-35-fold increase in dTMP incorporation opposite T (T----A transversion); these results are consistent with parallel genetic fidelity measurements. DNA polymerase beta also produces base substitution errors by direct misinsertion. Here nucleotide insertion fidelity results from substantial differences in both Km and Vmax for correct versus incorrect substrates and is influenced strongly by local base sequence.
Assuntos
DNA Polimerase II/metabolismo , DNA Polimerase I/metabolismo , DNA/genética , Mutação , Animais , Composição de Bases , DNA/metabolismo , Desoxicitidina Monofosfato/metabolismo , Desoxirribonucleotídeos/metabolismo , Drosophila/enzimologia , Cinética , Neoplasias Hepáticas Experimentais/enzimologia , Ratos , Saccharomyces cerevisiae/enzimologia , Moldes Genéticos , Timidina Monofosfato/metabolismo , Nucleotídeos de Timina/metabolismoRESUMO
The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.
Assuntos
DNA Polimerase I/metabolismo , Oligodesoxirribonucleotídeos/síntese química , Nucleotídeos de Timina/síntese química , Alquilação , Sequência de Bases , DNA/biossíntese , Indicadores e Reagentes , Cinética , Metilação , Relação Estrutura-Atividade , Moldes GenéticosRESUMO
We have determined the three-dimensional structure of a non-selfcomplementary nonanucleotide duplex which contains an abasic (apyrimidinic) site in the centre, i.e. a deoxyribose residue opposite an adenosine. The majority of the base and sugar proton resonances were assigned by NOESY, COSY and 2DQF spectra in D2O and H2O. We have measured the initial slope of buildup of NOEs in NOESY spectra at very short mixing times (25 to 50 ms), and from these were able to establish interproton distances for the central part of the duplex. We propose a different strategy for proton-proton distance determinations which takes into account the observed variations in correlation times for particular proton-proton vectors. A set of 31 measured interproton distances was incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. Two structures were obtained which retain all aspects of a classical B DNA in which the unpaired adenine and the abasic deoxyribose lie inside the helix. We observe that the non-hydrogen bonded adenine is held well in the helix, the Tm of this base being the same as that of the A.T base pairs in the same duplex.
Assuntos
DNA , Algoritmos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido NucleicoRESUMO
Variation of nonexchangeable proton resonance chemical shifts for deoxycytidine and deoxy-adenosine as a function of protonation and imino tautomer formation has been determined. Protonation induces downfield shifts of proton resonances whereas formation of the rare imino tautomer induces upfield shifts. Titration curves are constructed on the basis of spectrophotometrically determined pK values. Excellent fit is obtained between theoretical titration curves and experimental data, which indicates that chemical shifts of base protons may be used to quantitatively determine the relative concentrations of either rare imino tautomeric conformations or protonated base forms. These data may be utilized as an aid in the elucidation of the nature of hydrogen bonding between mismatched base pairs in DNA oligomers containing cytosine or adenine residues. These data, in conjunction with the oligonucleotide study of Patel et al. [Patel, D. J., Kozlowski, S.A., Ikuta, S., & Itakura, K. (1984) Biochemistry 23, 3218-3226], have been used to rigorously argue the existence of a "protonated" adenine residue in the A-C mismatch. This structure allows reconciliation of the NMR solution data with crystallographic data [Hunter, W.N., Brown, T., Anand, N.N., & Kennard, O. (1986) Nature (London) 320, 552-555], which support the protonated base pair.
Assuntos
Composição de Bases , DNA , Conformação de Ácido Nucleico , Desoxirribonucleosídeos , Concentração de Íons de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética/métodos , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
Ribonucleoside and deoxyribonucleoside triphosphate pools have been measured in Escherichia coli infected with bacteriophage T4 DNA polymerase mutator, wild type, and antimutator alleles during mutagenesis by the base analogue 2-aminopurine. ATP and GTP pools expand significantly during mutagenesis, while CTP and UTP pools contract slightly. The DNA polymerase (gene 43) alleles and an rII lesion perturb normal dNTP pools more than does the presence of 2-aminopurine. We find no evidence that 2-aminopurine induces mutations indirectly by causing an imbalance in normal dNTP pools. Rather, it seems likely that, by forming base mispairs with thymine and with cytosine, 2-aminopurine is involved directly in causing bidirectional A.T in equilibrium G.C transitions. The ratios for 2-aminopurine deoxyribonucleoside triphosphate/dATP pools are 5-8% for tsL56 mutator and 1-5% for tsL141 antimutator and 43+ alleles. We conclude that the significant differences observed in the frequencies of induced transition mutations in the three alleles can be attributed primarily to the properties of the DNA polymerases with their associated 3'-exonuclease activities in controlling the frequency of 2-aminopurine.cystosine base mispairs.
Assuntos
2-Aminopurina/farmacologia , Adenina/análogos & derivados , Desoxirribonucleotídeos/análise , Escherichia coli/genética , Mutação , Ribonucleotídeos/análise , Fagos T/genética , Alelos , DNA Polimerase Dirigida por DNA/genéticaRESUMO
We have investigated the mechanism of bromouracil-induced transition mutations in vitro using synthetic DNA templates and purified T4 DNA polymerase. Evidence is presented for the occurrence of bromouracil-guanine base pairs in product DNA in the G x C----A x T pathway where guanine is present in the DNA template and bromouracil is present as the deoxynucleoside triphosphate substrate 5-bromodeoxyuridine triphosphate. This finding supports a widely known but as yet untested model proposed by Freese (Freese, E. (1959) J. Mol. Biol. 1, 87-105) in which bromouracil-guanine base pairs are intermediates in 5-bromodeoxyuridine-induced transition mutation pathways. We find that the newly formed B x G base pairs are proofread with an efficiency of 75-85% by the 3' -exonuclease of T4 polymerase. The insertion of bromouracil occurring in direct competition with cytosine deoxyribonucleotides opposite template guanine sites is 1.1 +/- 0.14% (mean +/- S.E.), and the misincorporation ratio, inc(B)/inc(C), is reduced 6-fold by the action of the proofreading exonuclease to 0.16 +/- 0.02% (mean +/- S.E.). A previous study by Trautner et al. (Trautner, T. A., Swartz, M. N., and Kornberg, A. (1962) Proc. Natl. Acad. Sci. U. S. A. 48, 449-455) suggested that, while template bromouracil stimulates incorporation of dGMP in the A x T----G x C transition mutation pathway, it may not be occurring exclusively by the pathway proposed by Freese. We concur with these earlier results, and, in addition, we find the surprising result that the 3'-exonuclease activity of wild-type T4 polymerase removes little or no incorporated dGMP on bromouracil-containing templates.
Assuntos
Bromouracila/farmacologia , Mutação , Monofosfato de Adenosina/metabolismo , Sequência de Bases , Bromodesoxiuridina/análogos & derivados , Bromodesoxiuridina/metabolismo , Citidina Trifosfato/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo , Fagos T/enzimologia , Moldes Genéticos , Timidina Monofosfato/metabolismoRESUMO
[3H]2-Aminopurine deoxyribonucleoside triphosphate and [32P]dATP were added exogenously at equimolar concentrations to washed HeLa cell nuclei both in the presence and absence of cell cytoplasm. The observed ratio of 2-aminopurine/adenine deoxyribonucleotide incorporation into DNA was about 12%, which is consistent with 2-aminopurine misinsertion frequencies measured in cell-free assays, for various DNA polymerases including alpha-polymerase from calf thymus, Escherichia coli polymerase I, and several mutant and wild type bacteriophage T4 polymerases. Based on the 12% 2-aminopurine/adenine misincorporation ratio, we propose that proofreading of replicating DNA is not occurring in HeLa nuclei, and that discrimination against 2-aminopurine incorporation is governed primarily by a 1.1 kcal/mol difference in free energy between 2-aminopurine.thymine and adenine.thymine base pairs rather than by properties attributable to either the mammalian DNA polymerase or HeLa cell nuclear replication apparatus.
Assuntos
Núcleo Celular/metabolismo , DNA de Neoplasias/genética , Células HeLa/metabolismo , Cromatografia Líquida de Alta Pressão , Replicação do DNA , DNA de Neoplasias/biossíntese , Humanos , CinéticaRESUMO
We address the question of whether substituting 2-aminopurine (APur) in place of adenine (Ade) in DNA can increase the frequency of base mispairing with cytosine. Using DNA polymerase alpha to measure the rates of inserting deoxycytidine and thymidine nucleotides in direct competition with each other for APur or Ade sites on synthetic copolymer DNA templates, we observe that the ratio of dCMP to dTMP insertion is increased by a factor of at least 230 when APur replaces Ade on a poly(dA) template and by a factor of 35 when APur replaces Ade on a poly(dC,dA) template. These data support the idea that APur.C base mispairs are directly involved in APur induction of A.T leads to G.C transition mutations. The observed misinsertion frequency of cytosine substituting for thymine opposite template APur sites is about 5%. This value is in excellent agreement with earlier predictions and measurements for APur.C heteroduplex-heterozygote frequencies in T4 bacteriophage in vivo.