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PubMed1993

Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog phosphonoacetic acid.

Mutations that conferred sensitivity to the pyrophosphate analog phosphonoacetic acid in bacteriophage T4 DNA polymerase were identified. The mutations were loosely clustered in four regions of the gene. As found for herpes simplex virus DNA polymerase, T4 mutations that altered sensitivity to phosphonoacetic acid also altered sensitivity to nucleotide analogs. Some of the T4 DNA polymerase mutations also altered the ability of the enzyme to translocate from one template position to the next and affected DNA replication fidelity. Kornberg (A. Kornberg, Science 163:1410-1418, 1969) envisioned a DNA polymerase active center which accommodates primer terminus and template DNAs and the incoming nucleotide. Some mutations identified on the basis of sensitivity to phosphonoacetic acid may be part of such an active center because single amino acid substitutions simultaneously alter several DNA polymerase functions.

Bacteriophage T4↗

PubMedDate not supplied

Alkylating agents and DNA polymerases.

Alkylating agents, for example nitrogen "mustards", are variably toxic, mutagenic, carcinogenic and teratogenic, but by mechanisms which have not been clearly established. In particular, the mechanisms both of their delayed toxic effects (which are primarily against dividing cells, in association with retardation of the rate of cell division, disruption of mitoses, and breakages and other abnormalities of chromosomes) and of their carcinogenic actions are not understood. The literature on the testing of thousands of analogues has demonstrated great variability of effects on the various cell biological phenomena, and no aspect of chemical structure or biochemical reactivity of these agents has been established as especially related to any particular effect. Here theories of the mechanisms of action of alkylating agents are reviewed and it is suggested that impairment of the functions of DNA polymerase complexes might contribute to some of the effects of alkylating agents. In particular, impairment of replicative fidelity of DNA during the S-phase could contribute to some of the mitotic and chromosomal effects, as well as to their carcinogenic and teratogenic potencies. Some aspects of testing the effects of alkylating agents on components of the DNA synthetic pathway are mentioned. Emphasis is given to consideration of the various relevant levels (conventional plasma/tissue; tissue/tumour cell cytoplasm; tumour cell cytoplasm/tumour cell nucleus and tumour nuclear karyoplasm/tumour chromatin] of the pharmacokinetics and pharmacodynamics of the agents and their metabolites.

Alkylating Agents↗

PubMed2001

Arg660Ser mutation in Thermus aquaticus DNA polymerase I suppresses T-->C transitions: implication of wobble base pair formation at the nucleotide incorporation step.

We examined the replication fidelity of an Arg660Ser (R660S) mutant of Thermus aquaticus DNA polymerase I (Taq pol I). In a forward mutation assay, R660S showed a marked reduction in T-->C transitions, one of the most frequent errors made by the wild-type enzyme. Steady-state kinetics showed that R660S discriminates against dGTP incorporation at a template T 13-fold better than the wild-type. R660S was also 3.2-fold less efficient than the wild-type at extending a T:dG mismatch. These results indicate that R660S has enhanced fidelity during incorporation and extension, which reduces its T-->C transition frequency. Interestingly, R660S also discriminated correct from incorrect nucleotides at the incorporation step of C:dATP, A:dATP, G:dATP and C:8-OH-dGTP mispairs 28-, 6.0-, 4.1- and 6.8-fold better, respectively, than the wild-type, although it may not always be as accurate as the wild-type at the extension step. A structural model suggests that Arg660 may participate in two interactions that influence fidelity; the guanidinium group of Arg660 might interact with the incoming guanine base at the major groove and it might compete for forming another interaction with the primer terminus. Substituting Arg with Ser may eliminate or alter these interactions and destabilize the closed complex with incorrect substrates. Our data also suggest that T:dGTP and C:dATP base pairs form 'wobble' structures at the incorporation step of Taq pol I.

Arginine↗

PubMed2002

Involvement of DNA polymerase beta in DNA replication and mutagenic consequences.

Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.

Animals↗

PubMed2000

Fidelity of nucleotide insertion at 8-oxo-7,8-dihydroguanine by mammalian DNA polymerase delta. Steady-state and pre-steady-state kinetic analysis.

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol delta preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K(m)). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol delta preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol delta replication fidelity at 8-oxoG depends upon contributions from K(m), K(d)(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.

Adenosine Triphosphate↗

PubMed1992

Amplimers with 3'-terminal phosphorothioate linkages resist degradation by vent polymerase and reduce Taq polymerase mispriming.

The 3'-->5' exonuclease activity of Vent, a thermostable polymerase from Thermococcus litoralis, enhances DNA replication fidelity but also diverts PCR primers (amplimers) from targeted amplification by degrading their 3' termini. We demonstrate that amplimers with a 3-base 3'-terminal mismatch can be efficiently truncated by Vent to prime DNA polymerizations that compete with the specific amplification reaction. However, amplimers with phosphorothioate bonds joining their 3'-terminal residues are resistant to degradation and demonstrate greatly enhanced priming specificity. Slight destabilization of base-pairing by phosphorothioate bond-linked residues also diminishes extension of mispaired 3' amplimer termini in Taq polymerase-mediated amplifications.

Bacterial Proteins↗

PubMed2001

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations.

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.

Amino Acid Sequence↗

PubMed1996

Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe.

The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.

Amino Acid Sequence↗

PubMed1985

Lack of miscoding properties of 7-(2-oxoethyl)guanine, the major vinyl chloride-DNA adduct.

Chloroethylene oxide, an ultimate carcinogenic metabolite of vinyl chloride, was reacted with poly(deoxyguanylate-deoxycytidylate); the nucleic acid base adducts, 7-(2-oxoethyl)guanine and 3,N4-ethenocytosine, were analyzed by reverse-phase high-performance liquid chromatography. Chloroethylene oxide-modified poly(deoxyguanylate-deoxycytidylate) was assayed as template in a replication fidelity assay with Escherichia coli DNA polymerase I, and the newly synthesized product was subjected to nearest-neighbor analysis. Misincorporation rates of deoxyadenosine monophosphate and thymidine monophosphate were found to increase with the level of template modification. About 80% of the mispairing events were located opposite minor cytosine lesions. 7-(2-Oxoethyl)guanine, the major adduct identified (greater than 98% of the adducts), did not miscode for either thymine or adenine, failing to support an earlier hypothesis that the cyclic hemiacetal form, O6,7-(1'-hydroxyethano)guanine, could, by analogy with O6-methyl- and O6-ethylguanine, simulate adenine. Our results indicate that direct miscoding of 7-(2-oxoethyl)-guanine may contribute only slightly to the induction of mutations by chloroethylene oxide or vinyl chloride.

Chromatography, High Pressure Liquid↗

PubMed2004

Geminin is targeted for repression by the retinoblastoma tumor suppressor pathway through intragenic E2F sites.

The geminin protein is a critical regulator of DNA replication. It functions to control replication fidelity by blocking the assembly of prereplication complexes in the S and G(2) phases of the cell cycle. Geminin protein levels, which are low in G(0)/G(1) and increase at the G(1)/S transition, are controlled through coordinate transcriptional and proteolytic regulation. Here we show that geminin is regulated transcriptionally by the retinoblastoma tumor suppressor (RB)/E2F pathway. Initially, we observed that the activation of RB led to the repression of geminin transcription. Conversely, Rb-null mouse embryonic fibroblasts have enhanced the expression of geminin relative to wild type mouse embryonic fibroblasts. Similarly, an acute loss of Rb in mouse adult fibroblasts deregulated geminin RNA and protein levels. To delineate the responsible regulatory motifs, luciferase reporter constructs containing fragments of the geminin promoter were generated. An analysis of the critical regulatory cis-acting elements in the geminin promoter indicated that intragenic E2F sites down-stream of the first exon were responsible for RB-mediated repression of geminin. The direct analysis of the endogenous geminin promoter revealed that these intragenic E2F sites are occupied by E2F proteins, and the mutation of these sites eliminates responsiveness to RB. Together, these data link the expression of geminin to the RB/E2F pathway and represent the first promoter analysis of this important regulator of DNA replication.

Animals↗

PubMed1999

Processive proofreading and the spatial relationship between polymerase and exonuclease active sites of bacteriophage phi29 DNA polymerase.

phi29 DNA polymerase is a multifunctional enzyme, able to incorporate and to proofread misinserted nucleotides, maintaining a very high replication fidelity. Since both activities are functionally separated, a mechanism is needed to guarantee proper coordination between synthesis and degradation, implying movement of the DNA primer terminus between polymerization and 3'-5' exonuclease active sites. Using single-turnover conditions, we have demonstrated that phi29 DNA polymerase edits the polymerization errors using an intramolecular pathway; that is, the primer terminus travels from one active site to the other without dissociation from the DNA. On the other hand, by using chemical tags, we could infer a difference in length of only one nucleotide to contact the primer strand when it is in the polymerization mode versus the editing mode. Using the same approach, it was estimated that phi29 DNA polymerase covers a DNA region of ten nucleotides, as has been measured in other polymerases using different techniques.

Bacillus Phages↗

PubMed1999

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae.

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.

DNA Polymerase II↗

PubMed1999

DNA repair systems in archaea: mementos from the last universal common ancestor?

DNA repair in the Archaea is relevant to the consideration of genome maintenance and replication fidelity in the last universal common ancestor (LUCA) from two perspectives. First, these prokaryotes embody a mix of bacterial and eukaryal molecular features. Second, DNA repair proteins would have been essential in LUCA to maintain genome integrity, regardless of the environmental temperature. Yet we know very little of the basic molecular mechanisms of DNA damage and repair in the Archaea in general. Many studies on DNA repair in archaea have been conducted with hyperthermophiles because of the additional stress imposed on their macromolecules by high temperatures. In addition, of the six complete archaeal genome sequences published so far, five are thermophilic archaea. We have recently shown that the hyperthermophile Pyrococcus furiosus has an extraordinarily high capacity for repair of radiation-induced double-strand breaks and we have identified and sequenced several genes involved in DNA repair in P. furiosus. At the sequence level, only a few genes share homology with known bacterial repair genes. For instance, our phylogenetic analysis indicates that archaeal recombinases occur in two paralogous gene families, one of which is very deeply branched, and both recombinases are more closely related to the eukaryotic RAD51 and Dmc1 gene families than to the Escherichia coli recA gene. We have also identified a gene encoding a repair endo/exonuclease in the genomes of several Archaea. The archaeal sequences are highly homologous to those of the eukaryotic Rad2 family and they cluster with genes of the FEN-1 subfamily, which are known to be involved in DNA replication and repair in eukaryotes. We argue that there is a commonality of mechanisms and protein sequences, shared between prokaryotes and eukaryotes for several modes of DNA repair, reflecting diversification from a minimal set of genes thought to represent the genome of the LUCA.

Amino Acid Sequence↗

PubMed2006

Simulating the effect of DNA polymerase mutations on transition-state energetics and fidelity: evaluating amino acid group contribution and allosteric coupling for ionized residues in human pol beta.

The control of the catalytic power and fidelity of DNA polymerases involves the complex combined effect of the protein residues, the Mg2+ ions, and the interaction between the DNA bases. In an attempt to advance the understanding of catalytic control, we analyze the effect of the protein residues, taking human DNA polymerase beta as a model system. Specifically, we examine the ability of different theoretical models to reproduce the effect of ionized residues on the transition state (TS) binding energy and the corresponding k(pol)/KD. We also explore the role of the Mg2+ ions in the binding and catalysis processes. The application of the microscopic linear response approximation (LRA) and the semimacroscopic PDLD/S-LRA methods to a benchmark of mutational studies produces a semiquantitative correlation and indicates that these methods can provide predictive power. However, pre-steady-state and steady-state kinetic studies currently available do not give a unique benchmark, owing principally to widely varying experimental conditions. We believe that a more uniform experimental benchmark is needed for further refinement of the theoretical models. The analysis of the correlation between the results obtained by a rigorous thermodynamic cycle and by simpler approximations indicates that the protein reorganization between the open, i.e., unbound, form and the closed form does not change the magnitude of the calculated mutational effects in a major way for the experimental data used in this study. The use of the PDLD/S-LRA group contributions allows us to construct energy-based correlation diagrams that can help toward understanding the coupling, i.e., transfer of information, between the base-binding and catalytic sites and to gain a deeper insight into the molecular basis of DNA replication fidelity. Our analysis suggests that the allosteric matrix obtained by subtracting the correlation matrix of the correct and incorrect base pairs should prove useful in exploring the information transfer occurring between the base-binding and catalytic sites. This type of treatment should be especially effective when coupled with structural studies of polymerase-DNA-base mispair ternary complexes and studies using polymerase double mutants. We discuss the potential of direct calculations of binding energy of the TS in a rational design of TS analogues and in drug design.

Allosteric Regulation↗

PubMed2001

Enhanced ribonucleotide incorporation by an O-helix mutant of Thermus aquaticus DNA polymerase I.

The O-helix of DNA polymerases has been implicated in substrate discrimination and replication fidelity. In this study, wild-type Thermus aquaticus DNA polymerase I (Taq pol I) and an O-helix mutant A661E was examined for their ability to discriminate between ribonucleotides and deoxyribonucleotides. Steady-state nucleotide extension kinetics were carried out using a template cytidine and each nucleotide dNTP and rGTP. Wild-type Taq pol I and A661E demonstrated similar Vmax and Km values for the correct nucleotide dGTP. However, A661E discriminated between incorrect and correct nucleotide less well than wild-type; discrimination was reduced by factors of 9.5-, 5.6- and 15-fold for dATP, dTTP and rGTP, respectively. These data suggest that A661E is efficient polymerases in the presence of the correct deoxynucleotide, dGTP, but it is impaired in ability to discriminate between correct and incorrect deoxyribonucleotides or between ribo- and deoxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E alters the interactions between the O-helix and the terminal two phosphate groups in the primer strand.

DNA Polymerase I↗

PubMed1986

GATC sequence and mismatch repair in Escherichia coli.

The Escherichia coli mismatch repair system greatly improves DNA replication fidelity by repairing single mispaired and unpaired bases in newly synthesized DNA strands. Transient undermethylation of the GATC sequences makes the newly synthesized strands susceptible to mismatch repair enzymes. The role of unmethylated GATC sequences in mismatch repair was tested in transfection experiments with heteroduplex DNA of phage phi 174 without any GATC sequence or with two GATC sequences, containing in addition either a G:T mismatch (Eam+/Eam3) or a G:A mismatch (Bam+/Bam16). It appears that only DNA containing GATC sequences is subject to efficient mismatch repair dependent on E. coli mutH, mutL, mutS and mutU genes; however, also in the absence of GATC sequence some mut-dependent mismatch repair can be observed. These observations suggest that the mismatch repair enzymes recognize both the mismatch and the unmethylated GATC sequence in DNA over long distances. The presence of GATC sequence(s) in the substrate appears to be required for full mismatch repair activity and not only for its strand specificity according to the GATC methylation state.

Bacteriophage phi X 174↗

PubMed1991

Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction.

We present a kinetic partitioning mechanism by which the highly efficient 3'----5' exonuclease activity of T7 DNA polymerase maximizes its contribution to replication fidelity with minimal excision of correctly base-paired DNA. The elementary rate constants for the proposed mechanism have been measured directly from single-turnover experiments by using rapid chemical quench-flow techniques. The exonuclease activity of T7 DNA polymerase toward single-stranded DNA is quite fast (kx greater than 700 s-1). This rapid exonuclease is restrained with double-stranded DNA by a kinetic partitioning mechanism that favors the binding of the DNA to the polymerase site to prevent the rapid degradation of matched DNA and yet allows selective removal of mismatched DNAs. Both matched and mismatched DNAs bind tightly to the polymerase site, with approximately equal affinities, Kdp = 20 and 10 nM, respectively. Selective removal of the mismatch is governed by the rate of transfer of the DNA from the polymerase to the exonuclease site (kp----x). The rapid excision of matched DNA is limited by a slow transfer rate (kp----x = 0.2 s-1) from the polymerase to the exonuclease site relative to the rate of polymerization [kp = 300 s-1; Patel et al. (1991) Biochemistry (first of three papers in this issue)]. Removal of mismatched DNA is facilitated by its faster transfer rate (kp----x = 2.3 s-1) to the exonuclease site relative to the slow rate of polymerization over a mismatch [kpi = 0.012 s-1; Wong et al. (1991) Biochemistry (second of three papers in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1996

Roles of the DNA mismatch repair genes in colorectal tumorigenesis.

A rapid increase in our understanding of the basic mechanisms involved in the genesis of colorectal cancer has occurred. Important factors include the activation of oncogenes, the inactivation of tumor suppressor genes and the inactivation of DNA repair systems. Hereditary non-polyposis colorectal cancer (HNPCC) has been linked to germ-line mutations that inactivate one allele of a DNA mismatch repair gene, which pre-disposes affected individuals to a high frequency of early-onset cancer. We have developed a cellular model to study HNPCC using the DNA mismatch repair-deficient colon cancer cell line HCT116 and corrected the genetic deficiency by chromosome transfer. Human chromosome 3 corrected all manifestations of DNA mismatch repair deficiency in the cell line and restored sensitivity to the toxic effects of certain DNA alkylating agents. Data suggest that the DNA mismatch repair genes not only participate in the recognition and repair of DNA mismatches during new strand synthesis but may also signal cell cycle arrest in the face of overwhelming DNA damage. Additional functions for the DNA mismatch repair genes in the maintenance of DNA replicative fidelity are being studied.

Alleles↗