Fidelity and processivity of DNA synthesis by DNA polymerase kappa, the product of the human DINB1 gene

Mammalian DNA polymerase kappa (pol kappa), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol kappa copies undamaged DNA with average single-base substitution and deletion error rates of 7 x 10(-3) and 2 x 10(-3), respectively. These error rates are high when compared to those of most other DNA polymerases. pol kappa also has unusual error specificity, producing a high proportion of T.CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol kappa can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol kappa is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.     

Picornavirus RNA-dependent RNA polymerase

Replication of the picornavirus genome is catalysed by a viral encoded RNA-dependent RNA polymerase, termed 3D polymerase. Together with other viral and host proteins, this enzyme performs its functions in the cytoplasm of host cells. The crystal structure of 3D polymerase from a number of picornaviruses has been determined. This review discusses the structure and function of the poliovirus 3D polymerase. The high error rates of 3D polymerase result in high sequence diversity such that virus populations exist as quasispecies. This phenomenon is thought to facilitate survival of the virus population in complex environments.     

The base substitution fidelity of DNA polymerase beta-dependent single nucleotide base excision repair

Damaged DNA bases are removed from mammalian genomes by base excision repair (BER). Single nucleotide BER requires several enzymatic activities, including DNA polymerase and 5',2'-deoxyribose-5-phosphate lyase. Both activities are intrinsic to four human DNA polymerases whose base substitution error rate during gap-filling DNA synthesis varies by more than 10,000-fold. This suggests that BER fidelity could vary over a wide range in an enzyme dependent manner. To investigate this possibility, here we describe an assay to measure the fidelity of BER reactions reconstituted with purified enzymes. When human uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and DNA ligase 1 replace uracil opposite template A or G, base substitution error rates are 相似文献   

Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice

RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirus-receptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase.     

PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases.

The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6)) < < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2-3 mM MgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions, the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) than the error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfu increased approximately 9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow, suggesting that the parameters which influence replication error rates may be similar in pol l- and alpha-like polymerases. Finally, the fidelity of 'long PCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but approximately 3-4-fold higher than the error rate of Pfu DNA polymerase.     

High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase.

We demonstrate that despite lacking a 3'----5' proofreading exonuclease, the Thermus aquaticus (Taq) DNA polymerase can catalyze highly accurate DNA synthesis in vitro. Under defined reaction conditions, the error rate per nucleotide polymerized at 70 degrees C can be as low as 10(-5) for base substitution errors and 10(-6) for frameshift errors. The frequency of mutations produced during a single round of DNA synthesis of the lac Z alpha gene by Taq polymerase responds to changes in dNTP concentration, pH, and the concentration of MgCl2 relative to the total concentration of deoxynucleotide triphosphates present in the reaction. Both base substitution and frameshift error rates of less than 1/100,000 were observed at pH 5-6 (70 degrees C) or when MgCl2 and deoxynucleotide triphosphates were present at equimolar concentrations. These high fidelity reaction conditions for DNA synthesis by the Taq polymerase may be useful for specialized uses of DNA amplified by the polymerase chain reaction.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).     

DNA polymerase alpha and models for proofreading.

Using a modified system to measure fidelity at an amber site in phi X174, we have employed DNA polymerase alpha to test different mechanisms for proofreading. DNA polymerase alpha does not exhibit the characteristics of "kinetic proofreading" seen with procaryotic polymerases. Polymerase alpha shows no evidence for a "next nucleotide" effect, and added deoxynucleoside monophosphates do not alter fidelity. Pyrophosphate, which increases error rates with a procaryotic polymerase, appears to weakly improve polymerase alpha fidelity. DNA polymerase alpha does exhibit a dramatic increase in error rate in the presence of a deoxycytidine thiotriphosphate (dCTP alpha S), but this enhanced mutagenesis also occurs under conditions where kinetic proofreading should be otherwise defeated. This particular effect with dCTP alpha S appears specific for DNA polymerase alpha and is not seen with the other polymerases tested.     

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.     

Purification and properties of spleen necrosis virus DNA polymerase.

DNA polymerase was purified to apparent electrophoretic homogeneity from virions of spleen necrosis virus (SNV). (SNV is a member of the reticuloendotheliosis group of avian ribodeoxyviruses). The SNV DNA polymerase appears to consist of a single polypeptide with a molecular weight of 68,000. The SNV DNA polymerase has a preference for Mn2+ for DNA synthesis with an RNA template and Mg2+ for DNA synthesis with a deoxyribohomopolymer template. At the optimum concentrations of divalent cation, the relative rates of DNA synthesis by SNV DNA polymerase with different template.primers were similar to the relative rates of DNA synthesis by an avian leukosis virus DNA polymerase, with the exception of a lower relative rate of DNA synthesis by SNV DNA polymerase with SNV RNA. However, in contrast to DNA synthesized by the avian leukosis virus DNA polymerase with a SNV RNA template, DNA synthesized by SNV DNA polymerase with an SNV RNA template did not hybridize to the SNV RNA. SNV DNA polymerase has RNase H activity which is antigenically distinct from the RNase H activity of avian leukosis-sarcoma virus DNA polymerase.     

Effect of 2'',3''-dideoxythymidine-5''-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.     

DNA polymerase mutagenic bypass and proofreading of endogenous DNA lesions

DNA polymerases differentiate between correct and incorrect substrates during synthesis on undamaged DNA templates through the biochemical steps of base incorporation, primer-template extension and proofreading excision. Recent research examining DNA polymerase processing of abasic, alkylation and oxidative lesions is reviewed in light of these discrimination mechanisms. Inhibition of DNA synthesis results from correct polymerase discrimination against utilization of geometrically incorrect template bases or 3' terminal basepairs. The efficiency of translesion synthesis is thus related to the physical structure of the lesion containing DNA. However, variations in enzyme structure and kinetics result in translesion synthesis efficiencies that are also dependent upon the DNA polymerase. With a low probability, polymerase misinsertion events create a 3' lesion terminus which is geometrically favored over the correct lesion basepair, resulting in mutagenic translesion synthesis. For example, both polymerase alpha and polymerase beta appear to require the formation of a stable 3' primer-template structure for efficient abasic site translesion synthesis. However, the enzymes differ as to the precise molecular make-up of the stable DNA structure, resulting in different mutational specificities. Similar mechanisms may be applicable to oxidative damage, where mutational specificities dependent upon the DNA polymerase also have been observed. In vitro reaction conditions also influence DNA polymerase processing of lesions. Using an in vitro herpes simplex virus thymidine kinase (HSV-tk) gene forward mutation assay, we demonstrate that high dNTP substrate concentrations affect the mutagenic specificity of translesion synthesis using alkylated templates. The exonuclease-deficient Klenow polymerase error frequency for G-->A transition mutations using templates modified by N-ethyl-N-nitrosourea (ENU) was four-fold higher at 1000 microM [dNTP], relative to 50 microM [dNTP], consistent with an increased efficiency of extension of the etO6G.T mispair. Moreover, the frequency of other ENU-induced polymerase errors was suppressed when polymerase reactions contained 50 microM dNTP, relative to 1000 microM dNTP. The efficiency of proofreading as a polymerase error discrimination mechanism reflects a balance between the competing processes of 3'-->5' exonuclease removal of mispairs and polymerization of the next correct nucleotide. Polymerases that are devoid of a proofreading exonuclease generally display enhanced abasic site translesion synthesis relative to proofreading-proficient enzymes. In addition, the proofreading exonucleases of Escherichia coli Pol I and T4 DNA polymerases have been found to remove mispairs caused by abasic sites and oxidative lesions, respectively, resulting in lowered polymerase error rates. However, the magnitude of the exonuclease effect is small (less than 10-fold), and highly dependent upon the DNA polymerase-exonuclease. We have studied proofreading exonuclease removal of alkylation damage in the HSV-tk forward assay. We observed no significant reduction in the magnitude of the mutant frequency vs. dose-response curves when N-methyl-N-nitrosourea or ENU-treated templates were used in exonuclease-proficient Klenow polymerase reactions, as compared to the exonuclease-deficient polymerase reactions. Thus, available data suggest that proofreading excision of endogenous lesion mispairs does occur, but the efficiency is dependent upon the lesion and the DNA polymerase-exonuclease studied.     

Error rate and specificity of human and murine DNA polymerase eta

We describe here the error specificity of mammalian DNA polymerase eta (pol eta), an enzyme that performs translesion DNA synthesis and may participate in somatic hypermutation of immunoglobulin genes. Both mouse and human pol eta lack intrinsic proofreading exonuclease activity and both copy undamaged DNA inaccurately. Analysis of more than 1500 single-base substitutions by human pol eta indicates that error rates for all 12 mismatches are high and variable depending on the composition and symmetry of the mismatch and its location. pol eta also generates tandem base substitutions at an unprecedented rate, and kinetic analysis indicates that it extends a tandem double mismatch about as efficiently as other replicative enzymes extend single-base mismatches. This ability to use an aberrant primer terminus and the high rate of single and double-base substitutions support the idea that pol eta may forego strict shape complementarity in order to facilitate highly efficient lesion bypass. Relaxed discrimination is further indicated by pol eta infidelity for a wide variety of nucleotide deletion and addition errors. The nature and location of these errors suggest that some may be initiated by strand slippage, while others result from additional mechanisms.     

Solution structure of a viral DNA polymerase X and evidence for a mutagenic function.

The African swine fever virus DNA polymerase X (ASFV Pol X or Pol X), the smallest known nucleotide polymerase, has recently been reported to be an extremely low fidelity polymerase that may be involved in strategic mutagenesis of the viral genome. Here we report the solution structure of Pol X. The structure, unique within the realm of nucleotide polymerases, consists of only palm and fingers subdomains. Despite the absence of a thumb subdomain, which is important for DNA binding in other polymerases, we show that Pol X binds DNA with very high affinity. Further structural analyses suggest a novel mode of DNA binding that may contribute to low fidelity synthesis. We also demonstrate that the ASFV DNA ligase is a low fidelity ligase capable of sealing a nick that contains a G-G mismatch. This supports the hypothesis of a virus-encoded, mutagenic base excision repair pathway consisting of a tandem Pol X/ligase mutator.     

A comparative evaluation of methods for isolation of RNA-directed DNA polymerase from cells in a reconstituted system.

We have compared the relative merits of several procedures for the isolation of RNA-directed DNA polymerase (EC 2.7.7.7.) from cells using a reconsituted model system consisting of a mixture of woolly monkey (simian) sarcoma virus and a cultured human lymphoblastoid cell line, NC-37. When the cell-virus mixture was gently disrupted and fractionated by differential centrifugation, most of the added polymerase was recovered associated with a particulate fraction obtained from the post-mitochondrial supernatant. Purification of the polymerase was best achieved starting from this fraction. The particulate fraction itself can be purified by gel filtration through a Sepharose 2 B column. This procedure did not significantly alter the composition of viral and cellular DNA polymerases. Whereas as little as 7.5 - 10(5) viral particles were sufficient for the detection of RNA-directed DNA polymerase activity, a minimum of about 10(11) particles were necessary for the isolation and unequivocal characterization of the enzyme from the cell-virus mixture by subcellular fractionation and chromatographic separation from cellular DNA polymerases. Purified RNA-directed DNA polymerase had the same primer-template characteristics, sedimentation properties, and immunological cross reactivity as the enzyme purified from density gradient-banded virions of simian sarcoma virus. Methods involving total extraction of the cell-virus mixture either by repeated freezing and thawing followed by detergent treatment or by Dounce homogenization and treatment with high salt and detergent failed to provide RNA-directed DNA polymerase free of cellular DNA polymerases. Because of this, low levels of cellular RNA-directed DNA polymerase may be missed when these approaches are used.     

The interaction of RNA polymerase II with non-promoter DNA sites.

Various complexes formed between purified RNA polymerase II and simian virus 40 DNA have been characterized with respect to rates of formation, rates of dissociation, and initial velocity of RNA synthesis. Two different types of complexes can form on intact DNA templates. One of these is formed rapidly, but is quite labile; the other forms more slowly, but is moderately stable once formed. The introduction of a single strand break into DNA leads to rapid and stable complex formation, and thus is expected to create the favored binding site. The observed properties of these complexes provide a general framework for describing the interactions of RNA polymerase II at non-promoter DNA sites. This framework appears to be similar to that established for Escherichia coli RNA polymerase interactions, suggesting that the fundamental mode of non-promoter DNA binding is similar for the bacterial, plant, and mammalian enzymes.