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At least 739 records · Page 41Linked to original sources
PubMed1994

Comparative study of mutagenesis by O6-methylguanine in the human Ha-ras oncogene in E. coli and in vitro.

Single residues of O6-methylguanine (O6-meG) were introduced into the first or second position of codon 12 (GGC; positions 12G1 or 12G2, respectively) or the first position of codon 13 (GGT; position 13G1) of the human Ha-ras oncogene in phage M13-based vectors. After transformation of E.coli, higher mutant plaque frequencies (MPF) were observed at 12G1 and 13G1 than at 12G2 if O6-alkylguanine-DNA alkyltransferase (AGT) had been depleted, while similar MPF were observed at all three positions in the presence of active AGT. Taken together, these observations suggest reduced AGT repair at 12G2. Kinetic analysis of in vitro DNA replication in the same sequences using E. coli DNA polymerase I (Klenow fragment) indicated that variation in polymerase fidelity may contribute to the overall sequence specificity of mutagenesis. By constructing vectors which direct methyl-directed mismatch repair to the (+) or the (-) strand and comparing the MPF values in bacteria proficient or deficient in mismatch repair and/or AGT, it was concluded that, while mutS-mediated mismatch repair did not remove O6-meG from O6-meG:C pairs, this repair mechanism can affect O6-meG mutagenesis by repairing G:T pairs generated through AGT-induced demethylation of O6-meG:T replication intermediates.

Base Sequence↗
PubMed2002

Effect of the O6 substituent on misincorporation kinetics catalyzed by DNA polymerases at O(6)-methylguanine and O(6)-benzylguanine.

Misincorporation at a DNA-carcinogen adduct may contribute to formation of mutations if a polymerase proceeds past the lesion, compromising fidelity, as in the G:C to A:T mutations caused by O(6)-alkylguanine. Replication of primer/templates containing guanine (G), O(6)-methylguanine (O(6)-MeG), or O(6)-benzylguanine (O(6)-BzG) was assessed using T7 DNA polymerase exo(-) (T7(-)) and HIV-1 reverse transcriptase (RT). The steady-state parameters indicated that T7(-) and RT preferentially incorporated dTTP opposite O(6)-MeG and O(6)-BzG. The incorporation efficiencies (k(cat)/K(m)) were less for O(6)-BzG than O(6)-MeG for both dCTP and dTTP insertion. Pre-steady-state analysis indicated that the product formed during the burst phase, i.e., the burst amplitude, differed significantly between the unmodified 24-mer/36-G-mer and the O(6)-alkylG-containing substrates. Extension of the O(6)-BzG-containing duplexes was much more difficult for both polymerases as compared to O(6)-MeG, except when RT easily extended the O(6)-BzG:T base pair. The for binding of dCTP or dTTP to a RT*DNA complex containing O(6)-MeG was 8-fold greater than for dNTP binding to a complex containing unmodified DNA. The for a RT*DNA complex containing O(6)-BzG was 50-fold greater. In conclusion, the bulkier O(6)-BzG is a greater block to polymerization by T7(-) and RT than is O(6)-MeG, but some polymerization does occur with an O(6)-BzG substrate. Pre-steady-state analysis indicates that neither dCTP nor dTTP insertion is strongly preferred during polymerization of O(6)-BzG-containing DNA, unlike the case of O(6)-MeG. These results and others regarding polymerase stalling opposite O(6)-MeG and O(6)-BzG are discussed in the following paper in this issue [Woodside, A. M., and Guengerich, F. P. (2002) Biochemistry 41, 1039-1050].

Antineoplastic Agents↗
PubMed2004

Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase.

The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease(-) (T7(-)). RT and T7(-) effectively bypassed N(2)-methyl(Me)G and readily extended primers but were strongly blocked by N(2)-ethyl(Et)G, N(2)-isobutylG, N(2)-benzylG, and N(2)-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7(-) showed a decrease of 10(3) in k(cat)/K(m) for dCTP incorporation opposite N(2)-MeG and a further large decrease opposite N(2)-EtG. Misincorporation frequency was increased 10(2)-10(3)-fold by a Me group and another approximately 10(3)-fold by an Et group. dATP was preferentially incorporated opposite bulky N(2)-alkylG molecules. N(2)-MeG attenuated the pre-steady-state kinetic bursts with RT and T7(-), and N(2)-EtG eliminated the bursts. Large elemental effects with thio-dCTP(alphaS) were observed with N(2)-EtG (6- and 72-fold decreases) but were much less with N(2)-MeG, indicating that the N(2)-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of K(d(dCTP)) and K(d(DNA)) and k(off) rates of DNA substrates from RT and T7(-) indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.

Alkylation↗
PubMed2002

Relationship between low-frequency aircraft noise and annoyance due to rattle and vibration.

A near-replication of a study of the annoyance of rattle and vibration attributable to aircraft noise [Fidell et al., J. Acoust. Soc. Am. 106, 1408-1415 (1999)] was conducted in the vicinity of Minneapolis-St. Paul International Airport (MSP). The findings of the current study were similar to those reported earlier with respect to the types of objects cited as sources of rattle in homes, frequencies of notice of rattle, and the prevalence of annoyance due to aircraft noise-induced rattle. A reliably lower prevalence rate of annoyance (but not of complaints) with rattle and vibration was noted among respondents living in homes that had been treated to achieve a 5-dB improvement in A-weighted noise reduction than among respondents living in untreated homes. This difference is not due to any substantive increase in low-frequency noise reduction of acoustically treated homes, but may be associated with installation of nonrattling windows. Common interpretations of the prevalence of a consequential degree of annoyance attributable to low-frequency aircraft noise may be developed from the combined results of the present and prior studies.

Aircraft↗
PubMed2002

Gene cloning and polymerase chain reaction with proliferating cell nuclear antigen from Thermococcus kodakaraensis KOD1.

The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 'KOD DNA polymerase'. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.

Cloning, Molecular↗
PubMed1981

Effect of polyamines on the fidelity of macromolecular synthesis.

Addition of polyamines to in vitro systems containing suboptimal concentrations of Mg2+ markedly stimulated protein and RNA synthesis. This stimulation is observed only within a marrow range of polyamine concentration. The extend of stimulation of RNA synthesis was dependent on assay conditions. Addition of spermidine to the wheat germ system not only stimulated poly(U) directed polyphenylalanine synthesis, but also reduced the misincorporation of leucine. MS2-coat protein synthesis, studied in an E.coli cell-free system using either one of the two glutamyl-tRNAs as the only source of glutamine, suggested that in the presence of spermidine, codon recognition by these two isoacceptor tRNA molecules was more stringent. From these results it is concluded that polyamines contribute to the specificity of codon/anticodon interactions and thereby increase the fidelity of protein synthesis.

Codon↗
PubMed1995

AANA journal course: update for nurse anesthetists--anesthesia simulation: a tool for learning and research.

High fidelity simulators are a new technology for training and research in anesthesiology. The simulators accurately replicate the anesthetist's entire work environment, including the patient, anesthesia machine, monitors, and drugs. The trainee is allowed to develop and refine his skills in anesthesia management in a safe and realistic environment. By tailoring the scenario, both basic and advanced management skills can be taught. Learners can be exposed to serious or uncommon anesthetic events which they may not otherwise see during their training. Videotaping and case logs provide unbiased recording of the session. Simulators also allow the use of new instructional techniques that are untenable in a real operating room, such as allowing critical incidents to develop fully to teach management skills or redoing cases that were poorly handled. Allowing the student to make critical errors, pausing the simulation to discuss management, or replaying the case with different management techniques are valuable teaching tools. Research studies in human performance, learning and cognition, and anesthesia crisis management training are currently underway and may produce improved training methods for novice and expert anesthesia practitioners alike.

Education, Nursing↗
PubMed2006

A hitchhiker's guide to survival finally makes CENs.

Most strains of the yeast Saccharomyces cerevisiae contain many copies of a 2-microm plasmid, a selfish autonomously replicating DNA that relies on two different mechanisms to ensure its survival. One of these mechanisms involves the high fidelity segregation of the plasmids to daughter cells during cell division, a property that is starkly reminiscent of centromeres. A new study reported in this issue (see Hajra et al. on p. 779) demonstrates that this high fidelity is achieved by the 2-microm plasmid, effectively recruiting the centromeric histone Cse4 from its host yeast cell to forge its own centromere and finally revealing how the 2-microm plasmid has survived in budding yeasts over millions of years.

Cell Division↗
PubMed2001

Accuracy of lesion bypass by yeast and human DNA polymerase eta.

DNA polymerase eta (Pol eta) functions in the error-free bypass of UV-induced DNA lesions, and a defect in Pol eta in humans causes the cancer-prone syndrome, the variant form of xeroderma pigmentosum. Both yeast and human Pol eta replicate through a cis-syn thymine-thymine dimer (TT dimer) by inserting two As opposite the two Ts of the dimer. Pol eta, however, is a low-fidelity enzyme, and it misinserts nucleotides with a frequency of approximately 10(-2) to 10(-3) opposite the two Ts of the TT dimer as well as opposite the undamaged template bases. This low fidelity of nucleotide insertion seems to conflict with the role of Pol eta in the error-free bypass of UV lesions. To resolve this issue, we have examined the ability of human and yeast Pol eta to extend from paired and mispaired primer termini opposite a TT dimer by using steady-state kinetic assays. We find that Pol eta extends from mispaired primer termini on damaged and undamaged DNAs with a frequency of approximately 10(-2) to 10(-3) relative to paired primer termini. Thus, after the incorporation of an incorrect nucleotide, Pol eta would dissociate from the DNA rather than extend from the mispair. The resulting primer-terminal mispair then could be subject to proofreading by a 3'-->5' exonuclease. Replication through a TT dimer by Pol eta then would be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

DNA Damage↗
PubMed1998

Replicable item-cluster subcomponents in the NEO Five-Factor Inventory.

The popular NEO Personality Inventory-Revised (NEO-PI-R) has a short form--the NEO Five-Factor Inventory (NEO-FFI)--that taps the five broad factors with fidelity and reliability. However, conventional scoring of this short form does not provide scores on more specific aspects of the broad-bandwidth factors. In this study, 13 item clusters were found to replicate across halves of a sample of self-descriptions by adults (N = 732). Thirteen factor-analytically derived scales were developed for the item clusters. The scales demonstrated reliability and factor structure comparable to that of the 30 facet scales of the NEO-PI-R. Correlation and multiple regression analyses showed that the content coverage of the 13 scales has high overlap with that of the NEO-PI-R facet scales, but that representation of some facet scales is more moderate. Archival and existing data involving the NEO-FFI can be easily rescored for these 13 subcomponents, increasing the researcher's gain of information from this convenient personality inventory.

Adult↗
PubMed1987

Prokaryotic DNA replication mechanisms.

The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology. In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase). DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity. DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers). Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves. Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

DNA Replication↗
PubMed2003

Enhancement of DNA, cDNA synthesis and fidelity at high temperatures by a dimeric single-stranded DNA-binding protein.

Bacterial single-stranded DNA-binding proteins (SSBs) are required for DNA replication and repair. We have over-expressed and purified the native form and two His-tagged fusions of the SSB from Thermus thermophilus (TthSSB). The three proteins were found as dimers in solution. They bound in vitro to single-stranded DNA specifically over a temperature range of 4-80 degrees C, and the wild-type protein could withstand incubation at 94 degrees C for 2 min. Addition of TthSSB to PCR halved the elongation time required for the DNA polymerases of T.thermophilus (Tth) and Pyrococcus furiosus (Pfu) to synthesise DNA fragments in PCRs. The presence of TthSSB increased the fidelity of the proof- reading-free DNA polymerase of T.thermophilus. TthSSB was also able to bind single-stranded RNA, allowing a dramatic enhancement of the reverse transcription activity of its cognate Tth DNA polymerase during cDNA synthesis.

Cloning, Molecular↗
PubMed2001

Haematopoietic stem cell ageing.

The question of whether haematopoietic stem cells age has raised considerable controversy, and has been re-opened recently, as a result of the growing interest in stem cells for transplantation and gene therapy. Studies have focused on the generation of different blood cell elements and the capacity for self-renewal; properties that characterize stem cells. Taken together, it appears that basal haematopoiesis is maintained throughout life, yet, the capacity to cope with haematological stress is decreased in advanced age. In principle, stem cells derived from aged donors can be used for autologous transplantation, when needed to recover basic haematopoiesis. However, patterns of T cell development are altered in ageing, and intervention to augment T cell response still needs to be considered. Current methods for expansion and maintenance of stem cells in vitro enable examination of stem cell potential for long-term expansion and function. A critical evaluation of the possible risks of replicative senescence and developmental changes in stem cells has become feasible. Ageing effects may relate to cell replication, cell migration and lymphoid differentiation. Understanding of the mechanisms underlying these processes will enable the fidelity of stem cell expansion and maintenance of their potential for long-term function.

Animals↗
PubMed2002

The property of DNA polymerase zeta: REV7 is a putative protein involved in translesion DNA synthesis and cell cycle control.

Translesion DNA synthesis (TLS) is an important damage tolerance system which rescues cells from severe injuries caused by DNA damage. Specialized low fidelity DNA polymerases in this system synthesize DNA past lesions on the template DNA strand, that replicative DNA polymerases are usually unable to pass through. However, in compensation for cell survival, most polymerases in this system are potentially mutagenic and sometimes introduce mutations in the next generation. In yeast Saccharomyces cerevisiae (S. cerevisiae), DNA polymerase zeta, which consists of Rev3 and Rev7 proteins, and Rev1 are known to be involved in most damage-induced and spontaneous mutations. The human homologs of S. cerevisiae REV1, REV3, and REV7 were identified, and it is revealed that the human REV proteins have similar functions to their yeast counterparts, however, a large part of the mechanisms of mutagenesis employing REV proteins are still unclear. Recently, the new findings about REV proteins were reported, which showed that REV7 interacts not only with REV3 but also with REV1 in human and that REV7 is involved in cell cycle control in Xenopus. These findings give us a new point of view for further investigation about REV proteins. Recent studies of REV proteins are summarized and several points are discussed.

Animals↗
PubMed2001

Functional interactions and signaling properties of mammalian DNA mismatch repair proteins.

The mismatch repair (MMR) system promotes genomic fidelity by repairing base-base mismatches, insertion-deletion loops and heterologies generated during DNA replication and recombination. This function is critically dependent on the assembling of multimeric complexes involved in mismatch recognition and signal transduction to downstream repair events. In addition, MMR proteins coordinate a complex network of physical and functional interactions that mediate other DNA transactions, such as transcription-coupled repair, base excision repair and recombination. MMR proteins are also involved in activation of cell cycle checkpoint and induction of apoptosis when DNA damage overwhelms a critical threshold. For this reason, they play a role in cell death by alkylating agents and other chemotherapeutic drugs, including cisplatin. Inactivation of MMR genes in hereditary and sporadic cancer is associated with a mutator phenotype and inhibition of apoptosis. In the future, a deeper understanding of the molecular mechanisms and functional interactions of MMR proteins will lead to the development of more effective cancer prevention and treatment strategies.

Animals↗
PubMed2000

Single 8-oxo-guanine and 8-oxo-adenine lesions induce marked changes in the backbone structure of a 25-base DNA strand.

Structural changes in a 25-base DNA strand, induced by single 8-oxo-guanine or 8-oxo-adenine substitutions, were shown by using Fourier transform-infrared spectroscopy with multivariate statistics. Pronounced differences were demonstrated between the parent and derivatives with respect to base interactions and changes in the phospho-deoxyribose backbone. The greatest degree of change in the backbone likely occurred immediately adjacent to the 8-oxo group, potentially altering the stereochemistry at a distance. The 8-oxo lesions, formed from reactive oxygen species (e.g., hydroxyl radicals), may appreciably alter the conformational properties of strands at the replication fork, thus affecting the selectivity of polymerases, the proofreading capability of repair enzymes, and the fidelity of the transcriptional machinery.

Adenine↗
PubMed2000

Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase iota.

Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.

Base Pair Mismatch↗