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Results for “replication fidelity”

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At least 433 records · Page 24Linked to original sources
PubMed1991

Evidence for horizontal gene transfer in Escherichia coli speciation.

After extracting more than 780 identified Escherichia coli genes from available data libraries, we investigated the codon usage of the corresponding coding sequences and extended the study of gene classes, thus obtained, to the nature and intensity of short nucleotide sequence selection, related to constraints operating at the nucleotide level. Using Factorial Correspondence Analysis we found that three classes ought to be included in order to match all data now available. The first two classes, as known, encompass genes expressed either continuously at a high level, or at a low level and/or rarely; the third class consists of genes corresponding to surface elements of the cell, genes coming from mobile elements as well as genes resulting in a high fidelity of DNA replication. This suggests that bacterial strains cultivated in the laboratory have been fixed by specific use of antimutator genes that are horizontally exchanged.

Amino Acids↗
PubMed1992

Analysis of the spectrum of mutations induced by the rad3-102 mutator allele of yeast.

The product of the RAD3 gene of Saccharomyces cerevisiae is required for mitotic cell viability and excision repair of UV-induced pyrimidine dimers. Certain rad3 mutant alleles (originally called rem1) increase the rates of both spontaneous mitotic recombination and mutation. The increase in mutation rates is not dependent upon the presence of the RAD6 error-prone pathway. The mutator phenotype suggests that the wild-type RAD3 gene product may be involved in the maintenance of fidelity of DNA replication in addition to its known role in excision repair. To investigate the role that RAD3 might play in mutation avoidance, we have utilized a well-characterized shuttle vector system to study the mutational spectrum occurring in rad3-102 strains and compare it to that seen in RAD3 strains. The results put constraints on the role that the rad-102 mutant gene product must play if the RAD3 protein is a component of the replication complex. Alternatively, the mutational spectrum is consistent with the hypothesis that the rad3-102 mutant protein interferes with postreplication mismatch repair.

Adenosine Triphosphatases↗
PubMed1982

Human lymphocytes resistant to 6-thioguanine increase with age.

Using an autoradiographic technique we determined the number of circulating lymphocytes that were resistant to 6-thioguanine and which were presumably mutants at the hypoxanthine--guanine phosphoribosyl transferase locus. The number in normal individuals was found to increase exponentially with age. The data suggest a relationship between mutagenesis and ageing, perhaps by way of a decline with age in the fidelity of DNA replication or repair.

Adolescent↗
PubMed1987

An ab initio molecular orbital study on the characteristics of 8-hydroxyguanine.

To investigate the mechanism by which the 8-hydroxyguanine residue in DNA affects the fidelity of DNA replication, the intrinsic properties of this modified base were investigated using an ab initio molecular orbital method. The most stable 8-hydroxyguanine form was revealed to be 6,8-diketo. The addition of an oxygen atom to the 8 position of a guanine base was shown to change the electrostatic potential of the molecule entirely and to give it a negative character. This effect may influence the local structure of 8-hydroxyguanine-containing DNA and the interaction with DNA polymerase, thereby resulting in infidelity of DNA replication.

Base Composition↗
PubMed1979

Dissipation-error tradeoff in proofreading.

Chemical proofreading systems, of the kind believed responsible for the extremely high fidelity of DNA replication, achieve minimum error probability (equal to the product of the error probabilities of the writing and proofreading stages) only in the limit of infinite energy dissipation. However, a considerable degree of proofreading can be obtained in less strongly driven systems, dissipation only 0.1-1 kT/step.

Chemical Phenomena↗
PubMed1980

The anticarcinogenic effect of magnesium.

Evidence is reviewed supporting a hypothesis of an anticarcinogenic effect of magnesium. Animal studies reported increased cancer in rodents maintained on magnesium deficient diets and a preventive effect for rodents fed excess levels of magnesium. Epidemiologic evidence is presented that magnesium levels in water, food, and air are inversely related to cancer mortality. Magnesium may also have a role in the reduced cancer mortality experienced by people living at high altitudes. Two biologically plausible mechanisms are considered by which magnesium could prevent carcinogenesis. Intracellular magnesium may enhance the fidelity of DNA replication or magnesium on the cell membrane may prevent changes which trigger the carcinogenic process.

Air↗
PubMed1995

Transgenic models for detection of mutations in tumors and normal tissues of rodents.

Transgenic rodents that contain easily retrievable target genes allow the rapid quantitation of mutations in any tissue from which DNA can be isolated. We are using the Stratagene Big Blue transgenic mouse system that contains a lacI target and an alpha lacZ reporter gene to study the parameters that affect mutations. We have evaluated a number of chemicals to determine mutant frequency (MF) in specific target tissues of C57Bl/6 and B6C3F1 mice. The correlation between mutagenesis and carcinogenesis in this system is excellent. For example, the liver carcinogen dimethylnitrosamine produces significant increases in MF in mouse liver, whereas the nonhepatocarcinogenic mutagen methylmethane sulfonate does not. We have also evaluated the induction of mutations by radiation and demonstrated that this system is suitable for the study of agents that produce deletion mutations. This system is also useful for studying changes in MF in developing tumors. We have used an initiation-promotion protocol to induce hepatocellular carcinomas, and we then measured MF in normal liver, tumors, and metastases from these mice. Animals initiated with diethylnitrosamine maintain an elevated MF in normal liver, even 1 year after initiation. This MF increases exponentially in developing liver tumors, possibly owing to a breakdown in the fidelity of DNA replication and DNA repair in tumors. This system offers a unique tool for the study of mutations induced in specific target tissues of rodents and should become an important assay for evaluating the mutagenic risk of drugs and chemicals.

Animals↗
PubMedDate not supplied

Mismatch repair and DNA damage signalling.

Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.

Animals↗
PubMed2005

Differential modulation of base excision repair activities during brain ontogeny: implications for repair of transcribed DNA.

DNA repair sustains fidelity of genomic replication in proliferating cells and integrity of transcribed sequences in postmitotic tissues. The repair process is critical in the brain, because high oxygen consumption exacerbates the risk for accumulation of oxidative DNA lesions in postmitotic neurons. Most oxidative DNA damage is repaired by the base excision repair (BER) pathway, which is initiated by specialized DNA glycosylases. Because the newly discovered Nei-like mammalian DNA glycosylases (NEIL1/2) proficiently excise oxidized bases from bubble structured DNA, it was suggested that NEILs favor repair of transcribed or replicated DNA. In addition, since NEILs generate 3'-phosphate termini, which are poor targets for AP endonuclease (APE1), it was proposed that APE1-dependent and independent BER sub-pathways exist in mammalian cells. We measured expression and activities of BER enzymes during brain ontogeny, i.e., during a physiologic transition from proliferative to postmitotic differentiated state. While a subset of BER enzymes, exhibited declining expression and excision activities, expression of NEIL1 and NEIL2 glycosylases increased during brain development. Furthermore, the capacity for excision of 5-hydroxyuracil from bubble structured DNA was retained in the mature rat brain suggesting a role for NEIL glycosylases in maintaining the integrity of transcribed DNA in postmitotic brain.

Aging↗
PubMed2006

Efficient inefficiency: biochemical "junk" may represent molecular bridesmaids awaiting emergent function as a buffer against environmental fluctuation.

The biochemical function of many parts of the genome, transcriptome, proteome, and interactome remain largely unknown. We propose that portions of these fundamental building blocks of life have no current biochemical function per se. Rather, sections of these "omes" may contribute to an inventory of biochemical parts and circuits that participate in the development of emergent functions. Low fidelity deoxyribonucleic acid replication, transcription, translation, and post-translational modification all represent potential mechanisms to produce an inventory of parts. Stochastic processes that influence the conformations of ribonucleic acid molecules and proteins may also contribute to potential biochemical inventory. Some components of the biochemical inventory may enable future adaptations, some may produce disease, and some may remain useless. The function of many of these components await discovery, not by science, but by evolution. While carrying such purposeless biochemical units may appear to dilute fitness by exacting a thermodynamic cost, we argue that net fitness becomes enhanced when considering the value for potential future innovations. One can envision components that intermingle, interact, and act out mock pathways, but in most cases remain molecular bridesmaids. Given sufficiently low thermodynamic cost, such stochastic cycling may persist until a markedly advantageous or cataclysmically disadvantageous trait emerges. Maladaptive screening and utilization of inventory content can lead to disease phenotypes, a process buffered and regulated in part by the heat shock protein and stress response network. Whereas failure of the ubiquitin pathway to recycle misfolded proteins has become increasingly recognized as a source of disease, protein misfolding may itself represent one step in a process that maximizes functional innovation through increasing proteomic diversity. Fractal correlates of these processes occur at the organizational level of cells and organisms. That the abnormal accumulation of units induces local collapse may serve to limit the extension of damage to the greater system at large. The immune and cognitive systems that selectively sample and prune environmental content may serve as additional portals for innovation.

Adaptation, Physiological↗
PubMed2004

Subunit protein-affinity isolation of Drosophila DNA polymerase catalytic subunit.

gfLittle is known at present about the biochemical properties of very large-sized Drosophila DNA polymerases. In a previous study, we tried to purify Drosophila pol. catalytic subunit from embryos through seven column chromatographies and study its biochemical properties. However, we failed to characterize it precisely because an insufficient amount of the enzyme was generated. In this report, we describe direct purification from Drosophila embryos to near homogeneity using Drosophila DNA polymerase second subunit (Drosophila pol. 2) protein-conjugated affinity column chromatography and characterization of the enzyme in detail. To our knowledge this is the first demonstration of native DNA polymerase purification with activity using a subunit protein-affinity column. We observed new characteristics of Drosophila pol. catalytic subunit as follows: Drosophila pol. catalytic subunit synthesized DNA processively in the presence of both Mn(2+) and Mg(2+) ions, but Mn(2+) inhibited the 3'-5' proofreading activity, thereby decreasing the fidelity of DNA replication by 50%.

Animals↗
PubMed1999

Detection of microsatellite instability in sporadic cardiac myxomas.

OBJECTIVE: Microsatellite instability (MIN) is an early event in DNA repair-deficient associated diseases and reflects an elevated mutation rate in the genome of neoplastic cells. Sporadic cardiac myxomas are the most common primary heart tumours and their aetiopathology remains obscure. This study investigates the incidence of MIN in sporadic cardiac myxomas as a possible genetic mechanism of tumour pathogenesis. METHODS: Eleven surgically excised sporadic cardiac myxomas were assessed for MI using twenty-two highly polymorphic microsatellite markers, located on a wide range of chromosomal arms. DNA was extracted from myxoma tissue specimens as well as the respective normal tissue and subjected to polymerase chain reaction. RESULTS: The microsatellite analysis revealed that seven myxoma specimens (64%) exhibited MIN in at least one marker. One tumour specimen exhibited evidence of MIN in four microsatellite markers, while the most frequently affected marker was D17S855 (27%), located on chromosome 17q. DISCUSSION: We have detected a considerable incidence of MIN in sporadic cardiac myxomas indicating that decreased fidelity in DNA replication and repair is common in these tumours. To the best of our knowledge this is the first report describing MIN in sporadic cardiac myxomas, as a possible pathogenetic mechanism of these rare neoplasms.

Adult↗
PubMed2006

Characterization of the "mismatch repairosome" and its role in the processing of modified nucleosides in vitro.

The process of postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by eliminating biosynthetic errors from newly synthesized DNA. In addition, MMR proteins are also involved in the processing of intermediates of mitotic and meiotic recombination and, in mammalian cells, play a role in DNA damage signaling. As mismatches cannot be induced in the DNA of living cells, the study of the molecular transactions during MMR is restricted to in vitro systems. This chapter describes the construction of heteroduplex substrates that can be used for DNA affinity purification of MMR protein complexes and for the study of the role of eukaryotic MMR proteins in the processing of modified nucleosides.

Animals↗
PubMed2004

Structural basis for substrate selection by t7 RNA polymerase.

The mechanism by which nucleotide polymerases select the correct substrate is of fundamental importance to the fidelity of DNA replication and transcription. During the nucleotide addition cycle, pol I DNA polymerases undergo the transition from a catalytically inactive "open" to an active "closed" conformation. All known determinants of substrate selection are associated with the "closed" state. To elucidate if this mechanism is conserved in homologous single subunit RNA polymerases (RNAPs), we have determined the structure of T7 RNAP elongation complex with the incoming substrate analog. Surprisingly, the substrate specifically binds to RNAP in the "open" conformation, where it is base paired with the acceptor template base, while Tyr639 provides discrimination of ribose versus deoxyribose substrates. The structure therefore suggests a novel mechanism, in which the substrate selection occurs prior to the isomerization to the catalytically active conformation. Modeling of multisubunit RNAPs suggests that this mechanism might be universal for all RNAPs.

Binding Sites↗
PubMed2004

Structures of mismatch replication errors observed in a DNA polymerase.

Accurate DNA replication is essential for genomic stability. One mechanism by which high-fidelity DNA polymerases maintain replication accuracy involves stalling of the polymerase in response to covalent incorporation of mismatched base pairs, thereby favoring subsequent mismatch excision. Some polymerases retain a "short-term memory" of replication errors, responding to mismatches up to four base pairs in from the primer terminus. Here we a present a structural characterization of all 12 possible mismatches captured at the growing primer terminus in the active site of a polymerase. Our observations suggest four mechanisms that lead to mismatch-induced stalling of the polymerase. Furthermore, we have observed the effects of extending a mismatch up to six base pairs from the primer terminus and find that long-range distortions in the DNA transmit the presence of the mismatch back to the enzyme active site, suggesting the structural basis for the short-term memory of replication errors.

Base Pair Mismatch↗
PubMed2002

Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis.

Although toxicants may initiate cell damage or stress, the cellular proteins that are involved in control of cell cycle and apoptosis are the final arbiters of cell fate. The biochemical pathways that restrain cell cycle transition and/or induce cell death after stress are known as cell cycle checkpoints. These checkpoints maintain the fidelity of DNA replication, repair, and division. Herein, select cell cycle checkpoint signaling pathways will be discussed and how different components of these pathways are regulated by exogenous and endogenous agents, with focus on the p53 tumor suppressor signaling. The p53 protein is known to play a key role in growth arrest and apoptosis after cell stress, primarily through its ability to regulate the transcription of select downstream target genes in the cell. Further elucidation of the signaling pathways that control growth arrest and apoptosis will continue to provide insights to the complex cellular responses to environmental toxicants.

Animals↗
PubMedDate not supplied

Mutation rate: some biological and biochemical considerations.

This article discusses ideas about the ways in which the high fidelity of DNA replication is achieved: base selection, exonucleolytic editing, and postreplicative proofreading. I also review possible mechanisms for the enhanced mutation rate associated with SOS induction. The concept of environmental control of mutation rate and other modes of genetic variation is also considered from the point of view that SOS induction is an example of "genetic revolution".

Base Composition↗
PubMed1996

Human dUTP pyrophosphatase: uracil recognition by a beta hairpin and active sites formed by three separate subunits.

BACKGROUND: The essential enzyme dUTP pyrophosphatase (dUTPase) is exquisitely specific for dUTP and is critical for the fidelity of DNA replication and repair. dUTPase hydrolyzes dUTP to dUMP and pyrophosphate, simultaneously reducing dUTP levels and providing the dUMP for dTTP biosynthesis. A high cellular dTTP: dUTP ratio is essential to avoid uracil incorporation into DNA, which would lead to strand breaks and cell death. We report the first detailed atomic-resolution structure of a eukaryotic dUTPase, human dUTPase, and complexes with the uracil-containing deoxyribonucleotides, dUMP, dUDP and dUTP. RESULTS: The crystal structure reveals that each subunit of the dUTPase trimer folds into an eight-stranded jelly-roll beta barrel, with the C-terminal beta strands interchanged among the subunits. The structure is similar to that of the E. coli enzyme, despite low sequence homology between the two enzymes. The nucleotide complexes reveal a simple and elegant way for a beta hairpin to recognize specific nucleic acids: uracil is inserted into a distorted antiparallel beta hairpin and hydrogen bonds entirely to main-chain atoms. This interaction mimics DNA base pairing, selecting uracil over cytosine and sterically precluding thymine and ribose binding. Residues from the second subunit interact with the phosphate groups and a glycine-rich C-terminal tail of the third subunit caps the substrate-bound active site, causing total complementary enclosure of substrate. To our knowledge, this is the first documented instance of all three subunits of a trimeric enzyme supplying residues that are critical to enzyme function and catalysis. CONCLUSIONS: The dUTPase nucleotide-binding sites incorporate some features of other nucleotide-binding proteins and protein kinases, but seem distinct in sequence and architecture. The novel nucleic acid base recognition motif appears ancient; higher order structures, such as the ribosome, may have evolved from a motif of this kind. These uracil-beta-hairpin interactions are an obvious way for peptides to become early coenzymes in an RNA world, providing a plausible link to the protein-DNA world. Within the beta hairpin, there is a tyrosine corner motif that normally specifies beta-arch connections; this tyrosine motif was apparently recruited to discriminate against ribonucleotides, more recently than the evolution of the beta hairpin itself.

Amino Acid Sequence↗