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PubMed2003

General concepts in full scale simulation: getting started.

UNLABELLED: Over the past decade, medical simulation has developed to a point where it now is poised to become ubiquitous in teaching curricula. Despite this experience, there is little up-to-date information to help new instructors and operators learn the general principles of simulation. The purpose of this article is to provide prospective simulation instructors with basic concepts and a practical approach to patient simulation. The main focus is on full-scale or high fidelity simulation. The article is intended to (a). prepare instructors to use full-scale simulation to educate students; (b). teach some of the complexities and terminology of simulation; (c). prepare for and complement the curriculum of a formal instructor course; and (d). teach the basic elements required to run a successful simulation. This article should be used as an adjunct to practical experience gained from using simulation units. IMPLICATIONS: Medical simulation replicates normal and abnormal physiology and pathology. It is a tool that is intended to increase experiential learning. Establishing a functional and useful simulation program involves many factors. This paper presents a detailed introduction to the concepts and methodology of simulation in medicine.

Anesthesiology↗
PubMed2005

Processivity clamp gp45 and ssDNA-binding-protein gp32 modulate the fidelity of bacteriophage RB69 DNA polymerase in a sequence-specific manner, sometimes enhancing and sometimes compromising accuracy.

Numerous studies of the impact of accessory proteins upon the fidelity of DNA synthesis have provided a complex and sometimes discordant picture. We previously described such an analysis conducted in vitro using various bacteriophage RB69 gp43 mutator DNA polymerases with or without the accessory proteins gp32 (which binds single-stranded DNA) plus gp45/44/62 (processivity clamp and its loaders). Mutations were scored at many sites in the lacZalpha mutation reporter sequence. Unexpectedly, the accessory proteins sometimes decreased and sometimes increased fidelity at a handful of specific sites. Here, we enlarge our analysis with one particular mutator polymerase compromised in both insertion accuracy and proofreading and also extend the analysis to reactions supplemented only with gp32 or only with gp45/44/62. An overall 1.56-fold increase in mutation frequencies was produced by adding single or multiple accessory proteins and was driven mainly by increased T(template)*G(primer) mispairs. Evidence was found for many additional sites where the accessory proteins influence fidelity, indicating the generality of the effect. Thus, accessory proteins contribute to the site-specific variability in mutation rates characteristically seen in mutational spectra.

Bacteriophage T4↗
PubMed2002

A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication.

DNA polymerases that initiate replication by protein-priming are able to catalyze terminal protein (TP)-primed initiation, the following transition steps and finally DNA-primed elongation. Therefore, their structures must be able to position sequentially both primers, TP and DNA, at a common binding site. For DNA-templated initiation, these DNA polymerases have to bind the origin of replication as template and TP as primer. It is likely that very precise interactions are required to position both TP and templating nucleotide at the polymerization active site. Such a specificity during TP-priming must rely on specific amino acids that must be evolutionarily conserved in this subfamily of DNA polymerases. By site-directed mutagenesis, we have analyzed the functional significance of Lys392 of phi29 DNA polymerase, immediately adjacent to the Kx3NSxYG motif, and specifically conserved among protein-primed DNA polymerases. During TP-primed initiation, mutations in this residue did not affect untemplated TP-dAMP formation, indicating that the interaction with the initiating nucleotide and TP were not affected, whereas the template-directed initiation activity was severely inhibited. Both mutant DNA polymerases had a wild-type-like (overall) DNA binding activity. We thus infer that residue Lys392 of phi29 DNA polymerase is important for the correct positioning of the templating nucleotide at the polymerization active site, a critical requirement during template-directed TP-priming at phi29 DNA origins. Consequently, mutation of this residue compromised the fidelity of the initiation reaction, not controlled by the 3'-5' exonuclease activity. During DNA-primed polymerization, the mutant polymerases showed a defect in translocation of the template strand. This translocation problem could be the consequence of a more general defect in the stabilization and positioning of a next templating nucleotide at the polymerization active site, during DNA-primed DNA synthesis.

Amino Acid Sequence↗
PubMed1995

Virus DNA packaging: the strategy used by phage lambda.

Phage lambda, like a number of other large DNA bacteriophages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is co-ordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the lambda DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric lambda DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN to complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endonuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNu1, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in lambda has aspects that differ from other lambda DNA transactions. Unlike the site-specific recombination system of lambda, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complex, and unlike the DNA replication system of lambda, the same protein machinery is used for both initiation and translocation during lambda DNA packaging.

Amino Acid Sequence↗
PubMed1986

Isolation of two genes that affect mitotic chromosome transmission in S. cerevisiae.

Two DNA sequences that reduce mitotic fidelity of chromosome transmission have been identified: MIF1 and MIF2. MIF1 is a unique sequence located on the right arm of chromosome XII that stimulates loss and recombination for both chromosomes V and VII when present in a high copy number plasmid. MIF1 is not essential for cell division but is necessary for the normal fidelity of chromosome transmission. MIF2 is a unique sequence located 15 cM distal to HIS6 on chromosome IX that induces a high frequency of chromosome VII loss and a lower frequency of chromosome V loss when present in high copy number; it has no effect on mitotic recombination. Disruption of the genomic MIF2 locus was lethal and cells lacking this function arrested division with a terminal phenotype characteristic of a block in DNA replication or nuclear division.

Aneuploidy↗
PubMed1995

Implementing assertive community treatment teams.

The Connecticut Department of Mental Health began creating assertive community treatment teams in 1987. The authors describe the approach taken by the department in defining the assertive community treatment model, in creating new assertive community treatment teams, and in monitoring the creation and functioning of these teams to ensure that fidelity to the assertive community treatment model is maintained. Assertive community treatment teams can be created even in the absence of funding for new staff by reconfiguring current community-based staff and by moving staff from state hospitals to the community. Preliminary data from a randomized trial in Connecticut comparing assertive community treatment with high-quality case management in areas with an array of community services indicated that the intended models were replicated, with variations in practice style across programs. Clients in assertive community treatment were in the hospital about half as often as clients in standard services and were also less likely to be without a permanent residence. Training and ongoing monitoring of assertive community treatment teams are necessary to detect practices that diverge from the intervention model so that corrective action can be taken.

Adult↗
PubMed1996

Mutational studies of human immunodeficiency virus type 1 reverse transcriptase: the involvement of residues 183 and 184 in the fidelity of DNA synthesis.

The high error rates characteristic of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) are a presumptive source of the viral hypermutability that impedes prevention and therapy of acquired immunodeficiency syndrome (AIDS). We have analyzed two mutants of HIV-1 RT by conducting a comparative study of the accuracy of DNA synthesis. Each mutant bears a single amino acid substitution adjacent to the two aspartic acid residues at positions 185 and 186 in the highly conserved DNA polymerase active site. The first mutant, Met 184-->Leu (M184L), displays a marked reduction in both misinsertion and mispair extension, suggesting a fidelity of DNA synthesis significantly higher than that of the wild-type HIV-1 RT. The second mutant, Tyr 183-->Phe (Y183F), shows a decrease in mispair extension with no significant change in misincorporation. Thus, the overall pattern of error-proneness of DNA synthesis is: wild-type HIV-1 RT > Y183F > M184L. Taken together, it is possible that residues 183 and 184 contribute to the low fidelity of DNA synthesis characteristic of the reverse transcriptases of HIV-1, HIV-2 and possibly, of other lentiviruses. Our observations may bear on the nature of potential mutations responsible for resistance to the nucleoside analogs used in chemotherapy of AIDS.

Base Sequence↗
PubMed2006

A unique error signature for human DNA polymerase nu.

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.

Amino Acid Sequence↗
PubMed2002

Error catastrophe in mutant mitochondria.

The error catastrophe theory of aging, proposed by Orgel in 1963, predicted a decrease in the fidelity of information transfer that accelerated as aging progressed, until properly functioning macromolecules could no longer be reliably made. The theory was extensively tested by comparing DNA polymerases, transfer RNAs, and proteins derived from aging versus young animals, but it did not prove to have general applicability to the process of aging. Recently, the heritable eye disorder progressive external ophthalmoplegia has been found to result from mutation of the gene encoding DNA polymerase gamma, which replicates mitochondrial DNA. The mutant form of the polymerase replicates DNA less accurately than the wild-type enzyme, providing an explanation for the accumulation of mutations in the mitochondrial DNA of patients with this disorder. The affected mitochondria appear to exhibit an age-dependent error catastrophe. It is possible that other genetic diseases might result in error catastrophes in mitochondria as well.

Aging↗
PubMed1981

Fidelity of mammalian DNA polymerases.

The fidelity of copying natural DNA in vitro with each of the three classes of eukaryotic DNA polymerases has been determined. DNA polymerases-beta and -gamma are highly inaccurate, catalyzing noncomplementary single-base substitution at a frequency between 1/3000 and 1/8000. DNA polymerase-alpha is substantially more accurate, with an error rate of 1/30,000. When the error rates of these DNA polymerases are considered in the context of the accuracy of DNA replicative processes in vivo, it seems likely that other factors must exist in mammalian cells which are involved in the accurate replication and maintenance of the genetic information.

Animals↗
PubMed1996

Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase.

OBJECTIVE: To measure the extent of HIV resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine) within the context of monotherapy and to assess the presence of the M184V substitution in the case of 3TC-resistant viruses. Whether the success of 3TC in clinical trials could be due, in part, to an increase in the fidelity of HIV reverse transcriptase conferred by the M184V substitution was also considered. METHODS: Two separate monotherapy studies were evaluated, one involving adults with CD4 counts > or = 300 x 10(6)/l, and the second involving children, some of whom had received antiretroviral treatment previously, while others were drug naive. Peripheral blood and plasma samples were collected regularly, and HIV isolation and determinations of drug median inhibitory concentration values were performed using umbilical cord mononuclear cells as targets. Amplification of the 184 mutation was performed by the polymerase chain reaction, using specific primer pairs. Fidelity determinations using purified, recombinant HIV reverse transcriptase derived from either wild-type virus or viruses that contained the 184V substitution were performed. RESULTS: Phenotypic resistance was detected in almost all subjects at times ranging from 8-20 weeks after initiation of therapy. The 184V substitution was usually detected prior to the occurrence of phenotypic resistance to 3TC. Fidelity determinations revealed that the 184V substitution conferred an approximately 5- to 10-fold increase in HIV reverse transcriptase fidelity. In addition, titres of patient sera tested for their ability to neutralize autologous sequential viral isolates were stabilized in patients receiving 3TC therapy as opposed to other drugs. CONCLUSIONS: Resistance to 3TC developed in virtually all subjects treated with this drug, and was associated with the appearance of an M184V mutation in HIV reverse transcriptase. The clinical benefit of 3TC therapy may be attributable in part to selection of viruses that are less able to replicate and mutate than the wild types.

Adult↗
PubMed2003

Reduction of stability of arabidopsis genomic and transgenic DNA-repeat sequences (microsatellites) by inactivation of AtMSH2 mismatch-repair function.

Highly conserved mismatch repair (MMR) systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination, and responding to various DNA lesions. Arabidopsis and other plants encode a suite of MMR protein orthologs, including MSH2, the constant component of various specialized eukaryotic mismatch recognition heterodimers. To study MMR roles in plant genomic stability, we used Arabidopsis AtMSH2::TDNA mutant SALK_002708 and AtMSH2 RNA-interference (RNAi) lines. AtMSH2::TDNA and RNAi lines show normal growth, development, and fertility. To analyze AtMSH2 effects on germ line DNA fidelity, we measured insertion-deletion mutation of dinucleotide-repeat sequences (microsatellite instability) at nine loci in 16 or more progeny of two to four different wild-type or AtMSH2-deficient plants. Scoring 992 total alleles revealed 23 (2.3%) unique and 51 (5.1%) total repeat length shifts ([+2], [-2], [+4], or [-4] bp). For the six longest repeat loci, the corresponding frequencies were 22/608 and 50/608. Two of four AtMSH2-RNAi plants showed similar microsatellite instability. In wild-type progeny, only one unique repeat length allele was found in 576 alleles tested. This endogenous microsatellite instability, shown for the first time in MMR-defective plants, is similar to that seen in MMR-defective yeast and mice, indicating that plants also use MMR to promote germ line fidelity. We used a frameshifted reporter transgene, (G)(7)GUS, to measure insertion-deletion reversion as blue-staining beta-glucuronidase-positive leaf spots. Reversion rates increased only 5-fold in AtMSH2::TDNA plants, considerably less than increases in MSH2-deficient yeast or mammalian cells for similar mononucleotide repeats. Thus, MMR-dependent error correction may be less stringent in differentiated leaf cells than in plant equivalents of germ line tissue.

Arabidopsis↗
PubMed2004

Herpes simplex virus-1 primase: a polymerase with extraordinarily low fidelity.

We utilized templates of defined sequence to investigate the fidelity and mechanism of NTP misincorporation by DNA primase from herpes simplex virus-1. Herpes primase generated a wide range of mismatches during primer synthesis, including purine-purine, pyrimidine-pyrimidine, and purine-pyrimidine mismatches, and could even polymerize consecutive incorrect NTPs. Polymerization of noncognate NTPs resulted from primase misreading the template, as opposed to a primer slippage or dislocation mutagenesis mechanism. Primase did not efficiently misincorporate NTPs during the initiation reaction (i.e., dinucleotide synthesis). However, during primer elongation (after dinucleotide formation), herpes primase was extraordinarily inaccurate. It misincorporated NTPs at frequencies as high as 1 in 7, although frequencies of 1 in 25 to 1 in 60 were more common. In every case, however, misincorporation frequencies were no less than 1 in 100. For a specific mismatch, the DNA sequences flanking the site where misincorporation occurred could influence the frequency of misincorporation. This remarkably low level of fidelity is as low as that observed for the least accurate members of the Y class DNA polymerases involved in lesion bypass. Thus, herpes primase is one of the least accurate nucleotide polymerizing enzymes known.

Base Pair Mismatch↗
PubMed1976

Decreased fidelity of DNA polymerase activity isolated from aging human fibroblasts.

DNA polymerase (deoxynucleosidetriphosphate: DNA nucleotidyltransferase, EC 2.7.7.7 or DNA nucleotidyltransferase) activity, isolated from late and early passage cells of the diploid human fibroblast line, MRC-5, was compared. The level of activity dropped with increasing passage. In addition, when the fidelity of polymerization was monitored with four synthetic templates under a variety of conditions, it was observed that the enzyme from late passage cells was more error-prone. The possible relation of these observations to "senescence" of the fibroblasts is discussed.

DNA Nucleotidyltransferases↗
PubMed1996

Tissue regeneration as the basic oncogenic factor.

A new hypothesis of oncogenesis has been suggested. The errors of deoxyribonucleic acid (DNA) synthesis are supposed to be the main cause of oncogenic mutations. Since cell division and consequently DNA synthesis are continually needed for the renewal of some tissues in adult organisms, the physiological and reparative regeneration of these tissues (epithelium, blood) is the basic factor of oncogenesis. The exogenous and endogenous agents increase the probability of tumor frequency via increasing the cell renovation rate in tissues and decreasing the DNA synthesis fidelity. This hypothesis is in accord with the experimental and epidemiological data including the results which are not explained reasonably until now, namely, the association of the carcinogenic activity of drugs with their toxicity, the organ and tissue specificity of tumor frequency, etc.

Animals↗
PubMed2000

Fidelity and processivity of DNA synthesis by DNA polymerase kappa, the product of the human DINB1 gene.

Mammalian DNA polymerase kappa (pol kappa), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol kappa copies undamaged DNA with average single-base substitution and deletion error rates of 7 x 10(-3) and 2 x 10(-3), respectively. These error rates are high when compared to those of most other DNA polymerases. pol kappa also has unusual error specificity, producing a high proportion of T.CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol kappa can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol kappa is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.

Base Pair Mismatch↗
PubMed2026

Selective Inhibition of DNA Polymerase Proofreading: A Metabolic-Fidelity Mechanism Explains Agent Orange-Associated Myelodysplasia.

We performed a focused review to better understand the pathogenesis of Agent Orange (AO)-associated myelodysplastic syndrome (MDS). We first examined the mechanisms underlying conventional (de novo) MDS, a clonal hematopoietic neoplasm that typically develops in later life, and integrated these findings with our recent analysis of obesity-associated carcinogenesis. Accordingly, we propose that genomic instability in de novo MDS results from selective inhibition of the DNA polymerase proofreading exonuclease. In obesity-associated carcinogenesis, impaired AMP-activated protein kinase (AMPK) activity disrupts mitochondrial ATP production, increasing intracellular AMP concentrations. Elevated AMP selectively inhibits the proofreading exonuclease while preserving polymerase activity, allowing replication errors to escape correction and become fixed as somatic mutations. Molecular studies demonstrate that AO-associated MDS exhibits essentially the same mutational profile as de novo disease despite arising after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in young, otherwise healthy military personnel. Because TCDD is highly lipophilic, it accumulates in adipose tissue and is released slowly over decades, producing sustained mitochondrial dysfunction, reduced ATP synthesis, and chronic elevation of intracellular AMP. We propose that this metabolic disturbance converges on the same endpoint-selective inhibition of the proofreading exonuclease-thereby promoting mutagenesis and clonal evolution. Recent studies further strengthen the central role of proofreading by demonstrating that many mutations, including many found in MDS, previously attributed to spontaneous cytosine deamination, instead arise from DNA polymerase misincorporation of thymidine opposite cytosine, particularly at CpG dinucleotides, emphasizing the critical importance of fully active proofreading in preventing such misincorporations from accumulating as mutations in the genome of the cell.

AMP↗
PubMed2006

The 'ghosts' that pester studies on learning in mosquitoes: guidelines to chase them off.

The identification of memory and learning in medically important mosquito species has been of epidemiological interest mainly because of the implications of learning on the pattern of contact between vectors and hosts. Empirical results either showing or suggesting the existence of cognitive abilities in mosquitoes have been reported in a number of experimental studies, mainly based on the observation of individual fidelity towards subsets of specific resources, such as hosts, resting sites or breeding sites. A closer inspection of the design of these experiments shows that, with the exception of recent studies providing stronger evidence of learning in the genus Culex (Diptera: Culicidae), methodological shortcomings still hinder the possibility of eliminating alternative interpretations for these findings, in some cases because the experiments were not specifically designed to identify the phenomenon, but mostly because of a lack of appropriate controls or replication. By highlighting these limitations, while acknowledging the practical difficulties that are inherent to the field, we aim to help expel from future research the 'ghosts' that still preclude the achievement of more definite conclusions about the prevalence of memory and learning in this group of insects.

Animals↗