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At least 973 records · Page 54Linked to original sources
PubMed2000

Sloppier copier DNA polymerases involved in genome repair.

When chromosomal replication is impeded in the presence of DNA damage, members of a newly discovered UmuC/DinB/Rev1/Rad30 superfamily of procaryotic and eucaryotic DNA polymerases catalyze translesion synthesis at blocked replication forks. Although these polymerases share sequence elements essentially unrelated to the standard replication and repair enzymes, some of them (such as the SOS-induced Escherichia coli pol V) catalyze 'error-prone' translesion synthesis leading to large increases in mutation, whereas others (an example being the Xeroderma pigmentosum variant gene product XPV pol eta) carry out aberrant, yet nonmutagenic translesion synthesis. Ongoing studies of these low fidelity polymerases could provide new insights into the mechanism of somatic hypermutation, a key element in the immune response.

Animals↗
PubMed1994

A beta-like DNA polymerase from the mitochondrion of the trypanosomatid Crithidia fasciculata.

The mitochondrial DNA in Crithidia fasciculata, a trypanosomatid parasite, is known as kinetoplast DNA. Kinetoplast DNA has a very unusual structure, consisting of several thousand minicircles and a few dozen maxicircles, all topologically interlocked into a giant network. There is one network within each cell's single mitochondrion. We previously purified a 43-kDa DNA polymerase from C. fasciculata mitochondria (Torri, A. F., and Englund, P. T. (1992) J. Biol. Chem. 267, 4786-4792). This enzyme has properties very different from those of a DNA polymerase gamma, the conventional mitochondrial polymerase. In addition to its small size, it is nonprocessive, has no detectable exonuclease activity, and has very low fidelity. In all of these respects, the polymerase resembles a DNA polymerase beta, a gap-filling enzyme thought to function in DNA repair in the nucleus of other eukaryotes. We speculate that this enzyme may have been specially imported into the C. fasciculata mitochondrion to repair the many gaps found in minicircles following their replication. This is the first example of a beta-like polymerase from the mitochondrion of any eukaryote.

Amino Acid Sequence↗
PubMed1993

Analysis of mutations caused by DNA double-strand breaks produced by a restriction enzyme in shuttle vector plasmids propagated in ataxia telangiectasia cells.

Rejoining of DNA double-strand breaks (DSB) produced by a restriction endonuclease AvaI in the supF gene in a plasmid pZ189Ava, and mutations presumably due to the altered rejoinings were analyzed. After allowing the rejoining and replication of the plasmids in human cells originating from normal subjects and ataxia telangiectasia (AT) patients, the plasmids were retrieved and those containing mutated supF were screened in an indicator strain of Escherichia coli. The proportion of correctly rejoined plasmids was significantly lower in AT cells than in normal cells, suggesting that AT cells have lower fidelity in rejoining DSB. DNA sequencing of the mutated supF genes revealed that all mutations were deletions or insertions occurring exactly or closely at the rejoining site in both normal and AT cells. In AT cells, the majority of mutations were deletions, while deletions and insertions were evenly formed in normal cells. AT cells may be deficient in the mechanism to protect the broken ends of DNA strands from the exonucleolytic digestion.

Ataxia Telangiectasia↗
PubMed2004

Replicator-mutator equation, universality property and population dynamics of learning.

Replicator-mutator equation is used to describe the dynamics of complex adaptive systems in population genetics, biochemistry and models of language learning. We study "localized", or "coherent", solutions, which are especially relevant in the context of learning and correspond to the existence of a predominant language in the population. There is a coherence threshold for learning fidelity, above which coherent communication can be maintained. We prove the following surprising universality property of coherence threshold: for typical realizations of random coefficients in the fitness matrix, the value of the coherence threshold does not depend on the size of the system.

Biological Evolution↗
PubMed1997

Subcellular distribution of actively partitioning F plasmid during the cell division cycle in E. coli.

F plasmid is partitioned with fidelity to daughter cells during cell division cycle owing to two trans-acting genes, sopA and sopB, and a cis-acting site, sopC. We visualized the subcellular distribution of mini-F-plasmid molecules by fluorescence in situ hybridization. Mini-F-plasmid molecules having the sopABC segment were localized at midcell in newborn cells. Replicated plasmid molecules migrated to cell positions 1/4 and 3/4 without coupling with cell elongation and were tethered to these positions until completion of cell division. In contrast, molecules of a mini F plasmid lacking the sopABC segment were distributed randomly in spaces not occupied by nucleoids. The sopABC system caused replicated plasmid molecules to be positioned and tethered at the cell quarter sites.

Cell Division↗
PubMedDate not supplied

Immunonutrition: role of sulfur amino acids, related amino acids, and polyamines.

Pro-inflammatory cytokines mediate widespread changes in protein metabolism. Amino acids released from peripheral tissues fulfill a number of functions. They act as substrate for acute phase protein and immunoglobulin synthesis and, together with polyamines, in the replication of immune cells. Demands for specific amino acids may outstrip the supply from endogenous sources. A number of strands of evidence suggest that sulphur amino acids, and amino acids that are metabolically related to them, may be required in increased amounts. Protein deficiency impairs the acute phase response. However, sulfur amino acid insufficiency compromises glutathione synthesis, to a greater extent than hepatic protein synthesis, in the presence and absence of an inflammatory stimulus. The resulting effect may be compromised antioxidant defences. Functioning of T cells is dependent on intracellular glutathione concentrations and may also be affected by sulphur amino acid insufficiency. It has been suggested that the increased N excretion, which occurs during the immune response, is a reflection of a relative imbalance in the profile of amino acids released from peripheral tissues and the requirements imposed by the synthesis of substances involved in the acute phase response. Phenylalanine, tyrosine, tryptophan serine, and cysteine are released in amounts closest to requirements. Polyamine synthesis may be important for the fidelity of the enhanced level DNA transcription and RNA translation that occurs in response to infection and during tissue repair, gut growth after surgery, and in gut barrier functions. Although synthesized de novo from ornithine, arginine and S-adenosyl methionine (SAM), substantial recycling is a key feature of polyamine metabolism. The recycling may be a reflection of the need to maintain adequate tissue SAM during periods of rapid cell growth. During an immune/inflammatory response the combination of enhanced utilization of cysteine for glutathione synthesis and cell replication may lead to depletion of cellular SAM. A relatively small addition of polyamines to the diet may improve gut-associated aspects of the hosts' antibacterial defenses.

Amino Acids, Sulfur↗
PubMed1999

Mutational analysis of Phe160 within the "palm" subdomain of human immunodeficiency virus type 1 reverse transcriptase.

The highly conserved Phe160 residue is located in the "palm" subdomain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), and makes contact with Tyr115, a residue which is involved in deoxynucleoside triphosphate (dNTP) binding and fidelity of DNA synthesis. Five mutant RTs having Tyr, Trp, Ile, Ala or Gln instead of Phe160 were obtained by site-directed mutagenesis. F160Y and F160W retained substantial DNA polymerase activity, whereas the catalytic efficiency of nucleotide incorporation of mutants F160I, F160A and F160Q was less than 10 % that of the wild-type RT, using poly(rA).oligo(dT)20 as the template-primer. The low catalytic efficiency of mutants F160I, F160A and F160Q was due to their lower affinity for the dNTP substrate. F160Y displayed similar kinetic parameters as the wild-type RT in nucleotide insertion assays carried out with heteropolymeric DNA/DNA template-primers. However, nucleotide affinity was two- to sixfold reduced in the case of mutant F160W. Fidelity assays revealed similar misinsertion and mispair extension ratios for the three enzymes, although F160W showed a slightly higher accuracy of DNA synthesis, particularly in the presence of high concentrations of dNTP. When introduced in an infectious proviral clone, mutations F160I, F160A and F160Q rendered non-viable virus. The importance of Phe160 for polymerase function and viral replication could be mediated by its interaction with Tyr115, as suggested by the analysis of the available crystal structures of HIV-1 RT.

Amino Acid Substitution↗
PubMed2005

The role of viral fitness in HIV pathogenesis.

The development of clinical symptoms, and clinical progression among persons infected with HIV-1 is the manifestation of the effects of the pathogenic viral life cycle of HIV-1. Individual variants of HIV-1 vary widely in features that determine viral fitness and virulence. HIV-1 exploits host antiviral responses, the APOBEC3G cytidine deaminase, and the low-fidelity HIV-1 reverse transcriptase, to ensure new variants with novel phenotypic features are continually present for expansion in response to changing conditions in the host, such as immune responses, or antiretroviral therapy. This high-level variance has led to a wide range in observed fitness and virulence, across strains of HIV-1. The HIV-1 pol replication capacity assay (pol RC) measures features of viral fitness, associates with elevated CD4+ T-cell counts, yet is not strongly associated with HIV-1 RNA levels. The biological basis for elevated CD4+ T-cell counts among those carrying a virus of low pol RC may be because of lowered virus infectivity, or restricted tissue replication.

APOBEC-3G Deaminase↗
PubMed2004

Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase zeta.

Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase zeta (Pol zeta). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Pol zeta-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N(2)-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the gamma-hydroxy-1,N(2)-propano-2'deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N(2) of guanine. Acrolein, an alpha,beta-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Pol zeta subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N(2)-guanine DNA minor-groove adducts that form in DNA.

Acrolein↗
PubMed2006

DNA synthesis past a 5-methylC-containing cis-syn-cyclobutane pyrimidine dimer by yeast pol eta is highly nonmutagenic.

Cyclobutane pyrimidine dimers (CPDs) are responsible for a considerable fraction of sunlight-induced C to T and 5-methycytosine (mC) to T mutations in mammalian cells, though the precise mechanism is unknown. One possibility is that the C or mC of a CPD is not mutagenic and must first deaminate to U or T, respectively, for A to be inserted by a DNA polymerase. Alternatively, A might be directly inserted opposite the C or mC prior to deamination via an E-imino tautomer of the C or mC or by a nontemplated mechanism in which the photoproduct is sterically excluded from the active site. We have taken advantage of the retarding effect of C5 methylation on the deamination rate of cis-syn-cyclobutane dimers to prepare a template containing the cis-syn-cyclobutane dimer of mCT. Through the use of single-hit and multiple-hit competition assays, the catalytic core of pol eta was found to insert dGMP opposite the mC of the CPD with about a 120:1 selectivity relative to dAMP. No significant insertion of dTTP or dCMP was detected. The high fidelity of nonmutagenic insertion opposite the mC of the CPD provides strong support for the deamination-bypass mechanism for the origin of sunlight induced C --> T mutations.

DNA Replication↗
PubMed2004

Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition path sampling of DNA polymerase beta's closing.

Our application of transition path sampling to a complex biomolecular system in explicit solvent, the closing transition of DNA polymerase beta, unravels atomic and energetic details of the conformational change that precedes the chemical reaction of nucleotide incorporation. The computed reaction profile offers detailed mechanistic insights into, as well as kinetic information on, the complex process essential for DNA synthesis and repair. The five identified transition states extend available experimental and modeling data by revealing highly cooperative dynamics and critical roles of key residues (Arg-258, Phe-272, Asp-192, and Tyr-271) in the enzyme's function. The collective cascade of these sequential conformational changes brings the DNA/DNA polymerase beta system to a state nearly competent for the chemical reaction and suggests how subtle residue motions and conformational rate-limiting steps affect reaction efficiency and fidelity; this complex system of checks and balances directs the system to the chemical reaction and likely helps the enzyme discriminate the correct from the incorrect incoming nucleotide. Together with the chemical reaction, these conformational features may be central to the dual nature of polymerases, requiring specificity (for correct nucleotide selection) as well as versatility (to accommodate different templates at every step) to maintain overall fidelity. Besides leading to these biological findings, our developed protocols open the door to other applications of transition path sampling to long-time, large-scale biomolecular reactions.

DNA Polymerase beta↗
PubMed1993

Conformational coupling in DNA polymerase fidelity.

The fidelity of DNA polymerases is largely attributable to a two-step nucleotide binding mechanism. In the first step, binding contacts are initially made between the template and the incoming dNTP. The selectivity of this ground-state binding is similar in magnitude to the selectivity seen in forming base pairs in solution. In the second step, a change in protein conformation occurs, which leads to rapid incorporation of the dNTP into the growing polymer. This conformational change appears to occur globally in that it is inhibited by mismatches in the dNTP or in any of the three terminal base pairs of the primer/template. The open conformation allows rapid binding of the dNTP from solution, while the closed conformation provides steric checks for the proper Watson-Crick base pair geometry. This conformational change accounts for the extraordinary fidelity of polymerization and also provides selectivity to the exonuclease by inhibiting polymerization over a mismatch in the primer/template. The overall fidelity approaches one error in 10(10) by a combination of selectivity in polymerization (10(5)-10(6)) and in proofreading (10(3)-10(4)). This paradigm provides the theoretical basis for further investigation of the structural basis for fidelity by pointing to the essential elements of the polymerization reaction that need to be examined in order to evaluate active-site-directed mutants of polymerases to test appropriate structure/function relationships.

Base Sequence↗
PubMed2005

Chromatin architecture and functions: the role(s) of poly(ADP-RIBOSE) polymerase and poly(ADPribosyl)ation of nuclear proteins.

Epigenetic states that allow chromatin fidelity inheritance can be mediated by several factors. One of them, histone variants and their modifications (including acetylation, methylation, phosphorylation, poly(ADP-ribosyl)ation, and ubiquitylation) create distinct patterns of signals read by other proteins, and are strictly related to chromatin remodelling, which is necessary for the specific expression of a gene, and for DNA repair, recombination, and replication. In the framework of chromatin-controlling factors, the poly(ADP-ribosyl)ation of nuclear proteins, catalysed by poly(ADP-ribose)polymerases (PARPs), has been implicated in the regulation of both physiological and pathological events (gene expression/amplification, cellular division/differentiation, DNA replication, malignant transformation, and apoptotic cell death). The involvement of PARPs in this scenario has raised doubts about the epigenetic value of poly(ADP-ribosyl)ation, because it is generally activated after DNA damage. However, one emerging view suggests that both the product of this reaction, poly(ADP-ribose), and PARPs, particularly PARP 1, play a fundamental role in recruiting protein targets to specific sites and (or) in interacting physically with structural and regulatory factors, through highly reproducible and inheritable mechanisms, often independent of DNA breaks. The interplay of PARPs with protein factors, and the combinatorial effect of poly(ADPribosyl)ation with other post-translational modifications has shed new light on the potential and versatility of this dynamic reaction.

Adenosine Diphosphate Ribose↗
PubMed1999

Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the fission yeast Schizosaccharomyces pombe.

During meiosis homologous chromosomes replicate once, pair, experience recombination, and undergo two rounds of segregation to produce haploid meiotic products. The rec8(+), rec10(+), and rec11(+) genes of the fission yeast Schizosaccharomyces pombe exhibit similar specificities for meiotic recombination and rec8(+) is required for sister chromatid cohesion and homolog pairing. We applied cytological and genetic approaches to identify potential genetic interactions and to gauge the fidelity of meiotic chromosome segregation in the mutants. The rec8(+) gene was epistatic to rec10(+) and to rec11(+), but there was no clear epistatic relationship between rec10(+) and rec11(+). Reciprocal (crossover) recombination in the central regions of all three chromosomes was compromised in the rec mutants, but recombination near the telomeres was nearly normal. Each of the mutants also exhibited a high rate of aberrant segregation for all three chromosomes. The rec8 mutations affected mainly meiosis I segregation. Remarkably, the rec10 and rec11 mutations, which compromised recombination during meiosis I, affected mainly meiosis II segregation. We propose that these genes encode regulators or components of a "meiotic chromatid cohesion" pathway involved in establishing, maintaining, and appropriately releasing meiotic interactions between chromosomes. A model of synergistic interactions between sister chromatid cohesion and crossover position suggests how crossovers and cohesion help ensure the proper segregation of chromosomes in each of the meiotic divisions.

Aneuploidy↗
PubMed1995

Insights to primitive replication derived from structures of small oligonucleotides.

Available information on the structure of small oligonucleotides is surveyed. It is observed that even small oligomers typically exhibit defined structures over a wide range of pH and temperature. These structures rely on a plethora of non-standard base-base interactions in addition to the traditional Watson-Crick pairings. Stable duplexes, though typically antiparallel, can be parallel or staggered and perfect complementarity is not essential. These results imply that primitive template directed reactions do not require high fidelity. Hence, the extensive use of Watson-Crick complementarity in genes rather than being a direct consequence of the primitive condensation process, may instead reflect subsequent selection based on the advantage of accuracy in maintaining the primitive genetic machinery once it arose.

Base Composition↗
PubMed1997

Genomic instability in the type II TGF-beta1 receptor gene in atherosclerotic and restenotic vascular cells.

Cells proliferating from human atherosclerotic lesions are resistant to the antiproliferative effect of TGF-beta1, a key factor in wound repair. DNA from human atherosclerotic and restenotic lesions was used to test the hypothesis that microsatellite instability leads to specific loss of the Type II receptor for TGF-beta1 (TbetaR-II), causing acquired resistance to TGF-beta1. High fidelity PCR and restriction analysis was adapted to analyze deletions in an A10 microsatellite within TbetaR-II. DNA from lesions, and cells grown from lesions, showed acquired 1 and 2 bp deletions in TbetaR-II, while microsatellites in the hMSH3 and hMSH6 genes, and hypermutable regions of p53 were unaffected. Sequencing confirmed that these deletions occurred principally in the replication error-prone A10 microsatellite region, though nonmicrosatellite mutations were observed. The mutations could be identified within specific patches of the lesion, while the surrounding tissue, or unaffected arteries, exhibited the wild-type genotype. This microsatellite deletion causes frameshift loss of receptor function, and thus, resistance to the antiproliferative and apoptotic effects of TGF-beta1. We propose that microsatellite instability in TbetaR-II disables growth inhibitory pathways, allowing monoclonal selection of a disease-prone cell type within some vascular lesions.

Arteriosclerosis↗
PubMed2002

Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification.

The error rate of conventional PCR is problematic when amplifying from single cells or amplifying segments for protein functional analysis by in vitro translation. We describe truncated amplification, a method for high-fidelity amplification in which DNA polymerase errors are not propagated efficiently and original DNA templates exert greater influence on the amplification process. Truncated amplification utilizes pairs of oligonucleotides and thermal cycling, but it differs from PCR. Truncated amplification amplifies non-exponentially with one or two chimeric oligonucleotides and produces truncated terminal products that are no more than three rounds of replication from the original template. Exon 6 of the p53 gene was utilized as a model system to demonstrate proof of principle. Chimeric oligonucleotides containing three 3'-->5' reversed-deoxynucleotides or 2'-OMe-ribonucleotides at 6-8 nucleotides from the 3 'terminus retained sequence specificity and primer extension activity. With PfuTurbo but not with Taq or Vent (exo-) DNA polymerases, the modified nucleotides completely truncated the DNA polymerase elongation. The resulting truncated terminal products are not templates for further amplification because of the short length of the 3' complementary region. Truncated amplific ation can amplify quadratically or geometrically depending on whether two or one chimeric oligonucleotides are used. Truncated amplification is a promising approach when template-driven amplification is desired to increase thefrequency of error-free products.

Base Sequence↗
PubMed1989

Integration of mini-retroviral DNA: a cell-free reaction for biochemical analysis of retroviral integration.

After retroviral infection of a permissive cell, the viral RNA is reverse-transcribed to make a DNA copy of the viral genome. Integration of this DNA copy into the host genome is a necessary step for efficient viral replication. We have developed a cell-free system for integration of exogenous mini-retroviral DNA. The termini of this linear mini-Moloney murine leukemia virus (MoMLV) DNA are designed to mimic the ends of authentic unintegrated MoMLV DNA. The viral proteins required for integration can be provided either as a cytoplasmic extract of MoMLV-infected NIH 3T3 cells or as disrupted MoMLV particles. Phage lambda DNA serves as the target for integration. Genetic markers present on the mini-MoMLV DNA enable integration events to be detected, and the recombinants recovered, by selection in Escherichia coli. Integration, which occurs at heterogeneous locations in the target DNA, is absolutely dependent on the presence of a source of viral proteins and a divalent cation in the reaction mixture. The fidelity of the integration reaction was confirmed by sequencing the junctions between the integrated MoMLV DNA and adjacent lambda DNA sequence. In each case, as expected for authentic MoMLV DNA integration, a 4-base-pair duplication of target DNA sequence flanked the integrated MoMLV DNA.

Animals↗