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Results for “replication fidelity”

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At least 811 records · Page 45Linked to original sources
PubMed1992

Initiation of phi 29 DNA replication occurs at the second 3' nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication.

Bacteriophage phi 29 DNA replication is initiated when a molecule of dAMP is covalently linked to a free molecule of the terminal protein, in a reaction catalyzed by the viral DNA polymerase. We demonstrate that single-stranded DNA molecules are active templates for the protein-primed initiation reaction and can be replicated by phi 29 DNA polymerase. Using synthetic oligonucleotides, we carried out a mutational analysis of the phi 29 DNA right end to evaluate the effect of nucleotide changes at the replication origin and to determine the precise initiation site. The results indicate that (i) there are no strict sequence requirements for protein-primed initiation on single-stranded DNA; (ii) initiation of replication occurs opposite the second nucleotide at the 3' end of the template; (iii) a terminal repetition of at least two nucleotides is required to efficiently elongate the initiation complex; and (iv) all the nucleotides of the template, including the 3' terminal one, are replicated. A sliding-back model is proposed in which a special transition step from initiation to elongation can account for these results. The possible implication of this mechanism for the fidelity of the initiation reaction is discussed. Since all the terminal protein-containing genomes have some sequence reiteration at the DNA ends, this proposed sliding-back model could be extrapolable to other systems that use proteins as primers.

Bacillus Phages↗
PubMed2004

DNA polymerase gamma, the mitochondrial replicase.

DNA polymerase (pol) gamma is the sole DNA polymerase in animal mitochondria. Biochemical and genetic evidence document a key role for pol gamma in mitochondrial DNA replication, and whereas DNA repair and recombination were thought to be limited or absent in animal mitochondria, both have been demonstrated in recent years. Thus, the mitochondrial replicase is also apparently responsible for the relevant DNA synthetic reactions in these processes. Pol gamma comprises a catalytic core in a heterodimeric complex with an accessory subunit. The two-subunit holoenzyme is an efficient and processive polymerase, which exhibits high fidelity in nucleotide selection and incorporation while proofreading errors with its intrinsic 3' 5' exonuclease. Incorporation of nucleotide analogs followed by proofreading failure leads to mitochondrial toxicity in antiviral therapy, and misincorporation during DNA replication leads to mitochondrial mutagenesis and dysfunction. This review describes our current understanding of pol gamma biochemistry and biology, and it introduces other key proteins that function at the mitochondrial DNA replication fork.

Amino Acid Sequence↗
PubMedDate not supplied

Treatment fidelity plan for an activity intervention designed for persons with dementia.

The testing of psychosocial interventions in a clinical trial poses many challenges to maintaining a rigorous experimental protocol and to delivering the interventions uniformly throughout the project. These challenges directly affect the reported effectiveness of psychosocial interventions. In this article, the authors describe the treatment fidelity plan developed by an interdisciplinary research team from recreational therapy and nursing for implementing recreational activities during a clinical trial funded by the National Institute of Nursing Research. The trial tests the efficacy of activities for responding to the behavioral symptoms of dementia. The authors report treatment fidelity strategies to allow comparison of their intervention with that of other studies, to improve effect size in similar studies, and to facilitate replication and translation of this work into clinical practice.

Dementia↗
PubMed2006

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta.

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.

Catalysis↗
PubMed2004

Pre-steady-state kinetic studies of the fidelity of Sulfolobus solfataricus P2 DNA polymerase IV.

Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a thermostable archaeal enzyme and a member of the error-prone and lesion-bypass Y-family. In this paper, for the first time, the fidelity of a Y-family polymerase, Dpo4, was determined using pre-steady-state kinetic analysis of the incorporation of a single nucleotide into an undamaged DNA substrate 21/41-mer at 37 degrees C. We assessed single-turnover (with Dpo4 in molar excess over DNA) saturation kinetics for all 16 possible nucleotide incorporations. The fidelity of Dpo4 was estimated to be in the range of 10(-3)-10(-4). Interestingly, the ground-state binding affinity of correct nucleotides (70-230 microM) is 10-50-fold weaker than those of replicative DNA polymerases. Such a low affinity is consistent with the lack of interactions between Dpo4 and the bound nucleotides as revealed in the crystal structure of Dpo4, DNA, and a matched nucleotide. The affinity of incorrect nucleotides for Dpo4 is approximately 2-10-fold weaker than that of correct nucleotides. Intriguingly, the mismatched dCTP has an affinity similar to that of the matched nucleotides when it is incorporated against a pyrimidine template base flanked by a 5'-template guanine. The incoming dCTP likely skips the first available template base and base pairs with the 5'-template guanine, as observed in the crystal structure of Dpo4, DNA, and a mismatched nucleotide. The mismatch incorporation rates, regardless of the 5'-template base, were approximately 2-3 orders of magnitude slower than the incorporation rates for matched nucleotides, which is the predominant contribution to the fidelity of Dpo4.

Archaeal Proteins↗
PubMed1997

A genetic system to identify DNA polymerase beta mutator mutants.

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.

Animals↗
PubMed2006

EyeSim--an ophthalmic response simulator.

This paper pertains to the design, construction and development of an ophthalmic response simulator, intended to augment current neurological examination teaching practices in medical schools. Neurological examination of the eyes informs a practitioner of the functional state of certain cranial nerves (II, III, IV and VI) through the assessment of visual acuity, visual fields, ocular movements, external and internal eye structures and ophthalmoscopic examination. For the medical student, the interpretation of these indicators may be difficult, compounded by the infrequence with which such patients may be presented. As such, the need was identified for a simulator capable of replicating the ophthalmic response of patients with typical and a range of atypical neurologies. Such a simulator could be used as a stand-alone device or developed toward inclusion in pre-existing, life-size, hi-fidelity mannequins. Specifically, EyeSim will be able to track the movements of a pen-torch and vary the apparent dilation of an iris in response to changing levels of illumination. These responses can then be modulated to reflect certain deficits that neural pathologies may cause. At present the simulator comprises the electrical, electronic and mechanical components that replicate the pupillary response of the iris and the motility of the ocular muscles, a computer for the reception and analysis of a video signal and the micro-controller components, which determine the action of the physical components based on an array of inputs.

Computer Simulation↗
PubMed2002

Specialized DNA polymerases, cellular survival, and the genesis of mutations.

Cell death caused by arrested replication of damaged or structurally altered DNA can be avoided in prokaryotic and eukaryotic cells by multiple DNA polymerases that are specialized to bypass DNA damage. Some of these polymerases perform such translesion DNA synthesis of specific types of damage with high genetic fidelity. However, they exhibit greatly reduced fidelity when they operate on undamaged DNA or on DNA with lesions that are (apparently) not cognate substrates. The low fidelity of some of these specialized polymerases when copying undamaged DNA may be physiologically functional, including generating immunoglobulin diversity.

Animals↗
PubMed2002

Precocious S-phase entry in budding yeast prolongs replicative state and increases dependence upon Rad53 for viability.

Precocious entry into S phase due to overproduction of G1 regulators can cause genomic instability. The mechanisms of this phenomenon are largely unknown. We explored the consequences of precocious S phase in yeast by overproducing a deregulated form of Swi4 (Swi4-t). Swi4 is a late G1-specific transcriptional activator that, in complex with Swi6, binds to SCB elements and activates late G1-specific genes, including G1 cyclins. We find that wild-type cells tolerate Swi4-t, whereas checkpoint-deficient rad53-11 cells lose viability within several divisions when Swi4-t is overproduced. Rad53 kinase activity is increased in cells overproducing Swi4-t, indicating activation of the checkpoint. We monitored the transition from G1 to S in cells with Swi4-t and found that there is precocious S-phase entry and that the length of S phase is extended. Moreover, there were more replication intermediates, and firing of at least a subset of origins may have been more extensive in the cells expressing Swi4-t. Our working hypothesis is that Rad53 modulates origin firing based upon growth conditions to optimize the rate of S-phase progression without adversely affecting fidelity. This regulation becomes essential when S phase is influenced by Swi4-t.

Cell Cycle Proteins↗
PubMed2004

The impact of the M184V substitution on drug resistance and viral fitness.

Treatment of HIV/AIDS with antiretroviral therapy can result in HIV-1 drug resistance, limiting its use. Resistance mutations arise prior to therapy due to errors in HIV-1 replication, and are also spread by sexual and other modes of transmission. However, it is also generally believed that resistance is due to multiple drug mutations to any single or combination of antiretroviral agents selected during viral replication in the presence of incompletely suppressive drug regimens. In the case of protease inhibitors and most nucleoside analog reverse transcriptase inhibitors, drug resistance is due to the accumulation of mutations in the HIV-1 protease and reverse transcriptase genes respectively. However, in the case of non-nucleoside reverse transcriptase inhibitors, a single primary drug mutation is usually sufficient to abrogate antiviral activity. This is also true of certain specific mutations, such as M184V in the reverse transcriptase enzyme, resulting in resistance to the nucleoside analog, lamivudine (Epivir, GlaxoSmithKline). However, it is thought that lamivudine may still contribute to the effectiveness of antiretroviral therapy, even after the appearance of the M184V mutation. M184V may affect sensitivity to other drugs, such as zidovudine (Retrovir, GlaxoSmithKline), in HIV-1 variants that already contain resistance mutations to zidovudine, during concomitant treatment with lamivudine. M184V also has a positive effect on HIV-1 RT fidelity, reducing spontaneous HIV mutagenesis. Processivity of the reverse transcriptase enzyme may be affected by mutations such as M184V, and this may be a major determinant of viral replication fitness.

Amino Acid Substitution↗
PubMed2005

Adaptation to DNA damage and stimulation of genetic instability: the double-edged sword mammalian DNA polymerase kappa.

A major tolerance mechanism that functions to replicate damaged genomic DNA across lesions that have escaped elimination by repair mechanism is translesion DNA synthesis (TLS). DNA polymerase kappa (Pol kappa), a specialised low-fidelity DNA polymerase which is able to perform DNA synthesis across several damaged bases, is one of the enzymes involved in the process. The mutagenic nature of Pol kappa implies that its expression must be tightly regulated to prevent the formation of excessive genetic disorders along undamaged parts of the genome. Indeed, Pol kappa overexpression, which is notably observed in lung cancer, results not only in increased spontaneous mutagenesis, but also in pleiotropic alterations such as DNA breaks, genetic exchanges and aneuploidy. This review will discuss both aspects of DNA polymerase kappa, which can be considered as a genomic supervisor participating in genome maintenance and when misregulated as a genetic instability enhancer as well.

Animals↗
PubMed2004

Function of the C-terminus of phi29 DNA polymerase in DNA and terminal protein binding.

The thumb subdomain, located in various family B DNA polymerases in the C-terminal region, has been shown in their crystal structures to move upon binding of DNA, changing its conformation to nearly completely wrap around the DNA. It has therefore been involved in DNA binding. In agreement with this, partial proteolysis studies of phi29 DNA polymerase have shown that the accessibility of the cleavage sites located in their C-terminal region is reduced in the presence of DNA or terminal protein (TP), indicating that a conformational change occurs in this region upon substrate binding and suggesting that this region might be involved in DNA and TP binding. Therefore, we have studied the role of the C-terminus of phi29 DNA polymerase by deletion of the last 13 residues of this enzyme. This fragment includes a previously defined region conserved in family B DNA polymerases. The resulting DNA polymerase Delta13 was strongly affected in DNA binding, resulting in a distributive replication activity. Additionally, the capacity of the truncated polymerase to interact with TP was strongly reduced and its initiation activity was very low. On the other hand, its nucleotide binding affinity and its fidelity were not affected. We propose that the C-terminal 13 amino acids of phi29 DNA polymerase are involved in DNA binding and in a stable interaction with the initiator protein TP, playing an important role in the intrinsic processivity of this enzyme during polymerization.

Bacillus Phages↗
PubMed1985

Construction of telocentric chromosomes in Saccharomyces cerevisiae.

We describe a simple method for the construction of large chromosomal deletions in yeast. Diploid yeast cells were transformed with DNA fragments that replace large regions of the chromosomes by homologous recombination. Using this method, we have constructed a telocentric chromosome III in which approximately equal to 100 kilobases (kb) of DNA has been removed from the left arm of the chromosome, so that the centromere is 12 kb from the left telomere. This telocentric chromosome is mitotically stable. Its rate of loss in a diploid strain is 2.5-7.4 X 10(-4) per cell division compared to a rate of loss of 0.36-1.8 X 10(-4) per cell division for a normal chromosome III. It also segregates 2+:2- with fidelity during meiosis. The construction of systematic deletions in a chromosome should be useful in determining the essential features for proper chromosomal segregation and replication.

Base Sequence↗
PubMed1992

Alterations in nucleotide pools in rats fed diets deficient in choline, methionine and/or folic acid.

The fidelity of DNA synthesis is critically dependent on the correct balance and availability of the deoxynucleoside triphosphate (dNTP) precursors for the polymerases involved in replication and repair. Since folate-derived one-carbon groups are essential for the de novo synthesis of both purines and pyrimidines, the purpose of the present investigation was to determine whether diet-induced depletion of folates would alter intracellular dNTP pools. Fischer 344 rats were fed one of four semi-purified diets for a period of 8 weeks: (i) supplemented control; (ii) deficient in folic acid; (iii) deficient in methionine and choline; and (iv) deficient in methionine, choline and folic acid. In contrast to natural diets, semi-purified diets are nucleotide-free and consequently lack substrates for salvage pathway synthesis. This omission may place unusual stress on folate-dependent de novo nucleotide synthesis especially under conditions of dietary methyl-donor deficiency. Reversed-phase HPLC analysis of dNTP in spleen cell extracts indicated that both the thymidylate monophosphate and thymidylate triphosphate pools were decreased in spleen cells from the deficient rats consistent with a decrease in folate-dependent de novo synthesis. In addition, purine biosynthesis appeared to be negatively affect by methyl-donor deficiency as evidenced by a reduction in dGTP and dATP pools. These data indicate that deoxynucleotide pool imbalance, well known to produce cytogenetic and mutagenic events in vitro, can also be induced in this in vivo model of diet-induced carcinogenesis.

Animals↗
PubMed2003

Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR.

Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.

Binding Sites↗
PubMed2004

High mate and site fidelity in cunningham's skinks (Egernia cunninghami) in natural and fragmented habitat.

While habitat alteration has considerable potential to disrupt important within-population processes, such as mating and kin structure, via changed patterns of dispersal, this has rarely been tested. We are investigating the impact of anthropogenic habitat alteration on the population biology of the rock-dwelling Australian lizard Egernia cunninghami on the Central Tablelands of New South Wales, Australia, by comparing deforested and adjacent naturally vegetated areas. The novel analyses in this paper, and its companion, build on previous work by adding a new replicate site, more loci and more individuals. The additional microsatellite loci yield sufficient power for parentage analysis and the sociobiological inferences that flow from it. Genetic and capture-mark-recapture techniques were used to investigate mate and site fidelity and associated kin structure. Analyses of the mating system and philopatry using 10 microsatellite loci showed high levels of site fidelity by parents and their offspring in natural and deforested habitats. Parentage assignment revealed few individuals with multiple breeding partners within seasons and fidelity of pairs across two or more breeding seasons was typical. Despite reduced dispersal, increased group sizes and significant, dramatic increases in relatedness among individuals within rock outcrops in deforested areas, no significant differences between deforested and natural areas were evident in the degree of multiple mating or philopatry of breeding partners within and across seasons. With the exception that there was a significantly higher proportion of unmated males in the deforested area, the social and mating structure of this species has so far been surprisingly robust to substantial perturbation of dispersal and relatedness structure. Nonetheless, approximately 10-fold elevation of mean pairwise relatedness in the deforested areas has great potential to increase inbred matings, which is investigated in the companion paper.

Animals↗
PubMed2005

Translating a research intervention into community practice: the nurse family partnership.

Public policy initiatives have begun to recommend that interventions have strong evidence of effectiveness before there is expenditure of restrained public funds. The Nurse Family Partnership (NFP), a home visiting program for low-income parents expecting their first child, has been identified as a preventive intervention program that meets high evidentiary standards based on results from three randomized trials. Strategies used to promote successful translation of the research intervention into clinical practice, findings from the evaluation of the replication of the NFP in 22 states, and challenges experienced in moving a research program to practice are discussed. EDITORS' STRATEGIC IMPLICATIONS: Policymakers, community public health officials, and researchers planning to disseminate their prevention programs will find many lessons in this example of bringing a model program (i.e., a prevention strategy that works) up to scale. Although results at replication sites are somewhat weaker than at model sites, the consistent positive outcomes are a testimony to the strength of the NFP model and the fidelity of its implementation across sites.

Adult↗