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PubMed1985

Genetic analysis of the mitotic transmission of minichromosomes.

The fidelity of the mitotic transmission of minichromosomes in S. cerevisiae is monitored by a novel visual assay that allows one to detect changes in plasmid copy number in individual mitotic divisions. This assay is used to investigate the mitotic transmission of a plasmid containing a putative yeast origin of replication (ARS 1) and a centromere (CEN3). The rate of improper segregation for the minichromosome is 200-fold higher than observed for a normal chromosome. However, the replication of the minichromosome is stringently controlled; it overreplicates less than once per one thousand mitotic divisions. We also use this assay to isolate and characterize mutations in ARS 1 and CEN3. The mutations in ARS 1 define a new domain required for its optimal activity, and the mutations in CEN3 suggest that the integrity of element II is not essential for centromere function. Finally, the phenotypes of the mutations in ARS 1 and CEN3 are consistent with their function in replication and segregation, respectively.

Base Sequence↗

PubMed2003

S-phase checkpoint controls mitosis via an APC-independent Cdc20p function.

Cells divide with remarkable fidelity, allowing complex organisms to develop and possess longevity. Checkpoint controls contribute by ensuring that genome duplication and segregation occur without error so that genomic instability, associated with developmental abnormalities and a hallmark of most human cancers, is avoided. S-phase checkpoints prevent cell division while DNA is replicating. Budding yeast Mec1p and Rad53p, homologues of human checkpoint kinases ATM/ATR and Chk2, are needed for this control system. How Mec1p and Rad53p prevent mitosis in S phase is not known. Here we provide evidence that budding yeasts avoid mitosis during S phase by regulating the anaphase-promoting complex (APC) specificity factor Cdc20p: Mec1p and Rad53p repress the accumulation of Cdc20p in S phase. Because precocious Cdc20p accumulation causes anaphase onset and aneuploidy, Cdc20p concentrations must be precisely regulated during each and every cell cycle. Catastrophic mitosis induced by Cdc20p in S phase occurs even in the absence of core APC components. Thus, Cdc20p can function independently of the APC.

Anaphase-Promoting Complex-Cyclosome↗

PubMed1999

A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage.

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

Amino Acid Sequence↗

PubMed1978

Gene duplication in Saccharomyces cerevisiae.

Five independent duplications of the acid-phosphatase (aphtase) structural gene (acp1) were recovered from chemostat populations of S. cerevisiae that were subject to selection for in vivo hyper-aphtase activity. Two of the duplications arose spontaneously. Three of them were induced by UV. All five of the duplication events involved the transpositioning of the aphtase structural gene, acp1, and all known genes distal to acp1 on the right arm of chromosome II, to the terminus of an arm of other unknown chromosomes. One of the five duplicated regions of the right arm of chromosome II was found to be transmitted mitotically and meiotically with very high fidelity. The other four duplicated regions of the right arm of chromosome II were found to be unstable, being lost at a rate of about 2% per mitosis. However, selection for increased fidelity of mitotic transmission was effective in one of these strains. No tandem duplications of the aphtase structural gene were found.

Acid Phosphatase↗

PubMed1983

Accuracy of DNA polymerase-alpha in copying natural DNA.

The fidelity of DNA polymerase-alpha from calf thymus (9S enzyme) in copying bacteriophage phi174am16 DNA in vitro has been determined from the frequency of production of different revertants. In the self-priming reaction we were able to measure the frequencies of base pairing mismatches during the course of replication on biasing the ratios of deoxynucleoside triphosphates. The frequency of dGTP:T, dGTP:G and dATP:G mismatches were 7.6 x 10(-5), 4.4 x 10(-5) and 2.8 x 10(-5), respectively, at equal concentrations of the deoxynucleoside triphosphates. dCTP:A, dGTP:A, dCTP:T and dTTP:T mismatches were below the limit of detection (<5 x 10(-6)). A synthetic dodecamer primer with a 3' end covering the first two bases of the amber codon was used to determine the misinsertion frequency of the first nucleotide incorporated. This gave a misinsertion frequency of 1.5 x 10(-4) for the dGTP:T mismatch, which is slightly higher than that observed from the pool bias studies. Further, it showed no sensitivity to biasing the nucleotide pool, suggesting a different mechanism for the incorporation of the first nucleotide. These data do not support 'energy-relay'-like models for achieving high accuracy in eukaryotes. The observed misinsertion frequencies were corrected for mismatch repair of the heteroduplexes during the transfection experiments by parallel experiments using a mismatched primer. This was synthesized to have the same G:T mismatch as produced in the preceding experiment.

Animals↗

PubMed2003

Irreversible JPEG compression of digital chest radiographs for primary interpretation: assessment of visually lossless threshold.

PURPOSE: To determine if digital chest images could be compressed in a primary interpretation context without perceived loss of fidelity (below the visually lossless threshold) at transilluminated film or cathode ray tube (CRT) display. MATERIALS AND METHODS: One hundred forty-four posteroanterior radiographs were obtained with a digital chest radiography system. At both film and CRT display, an identified original image was presented side by side with a replicate, which was either an unaltered image or an image that had been Joint Photographic Experts Group (JPEG) compressed to 10:1, 20:1, or 50:1 and reconstructed. Each of the 10 readers indicated whether the replicate was "indistinguishable from the original" or "degraded" at clinical reading distance and at close inspection. The readers' ability to detect compressed images was examined for patterns; 95% CIs were used for statistical testing. RESULTS: With transilluminated film at clinical reading distance, readers were as likely to rate originals (48 [20%] of 240 readings) as degraded as they were to rate 20:1 replicates (106 [22%] of 480 readings) as degraded, but they frequently identified 50:1 replicates (283 [59%] of 480 readings) as degraded. At close inspection, 20:1 replicates (163 [34%] of 480 readings) were often identified as degraded, but 10:1 replicates (19 [8%] of 240 readings) were not identified as degraded more often than originals (17 [7%] of 240 readings). With CRT display, the results were nearly identical. CONCLUSION: At reading distance for primary interpretation, full-size digital chest radiographs that have been JPEG compressed to 10:1 or 20:1 and reconstructed are visually lossless at film or CRT display. Images compressed to 10:1 remain visually lossless at close inspection.

Algorithms↗

PubMed2005

Morphosynthesis: high fidelity inorganic replica of the fibrous network of loofa sponge (Luffa cylindrica).

High fidelity calcium carbonate and hydroxyapatite (bio) inorganic replicas of the fibrous network of the dried fruit of Luffa cylindrica are described, utilizing a facile synthetic route. The loofa sponge is a highly complex macroscopic architectural template, an inexpensive and sustainable resource. In the context of the morphosynthesis, the capability of replication of the loofa sponge opens the possibility of the use of biodiversity in obtaining new materials. We would like to emphasize that the template proposed in this paper, makes possible the preparation of inorganic replicas with a very desirable size, on the centimeter scale. This fact is innovative with respect to inorganic replicas described in the literature, which predominate at the micrometric scale, limited to the original size of the template.

Biomimetic Materials↗

PubMed1999

Abnormal, error-prone bypass of photoproducts by xeroderma pigmentosum variant cell extracts results in extreme strand bias for the kinds of mutations induced by UV light.

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C-->T, 30% of the base substitutions consist of C-->A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C-->T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZalpha, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to approximately 37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but approximately 2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C-->A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T-->A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand.

Cell Extracts↗

PubMed2000

Mechanisms of accurate translesion synthesis by human DNA polymerase eta.

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.

DNA Damage↗

PubMed2005

Application of Phi29 DNA polymerase mediated whole genome amplification on single spores of arbuscular mycorrhizal (AM) fungi.

Genetic analysis of arbuscular mycorrhizal (AM) fungi relies on analysis of single spores. The low DNA content makes it difficult to perform large scale molecular analysis. We present the application of Phi29 DNA polymerase mediated strand displacement amplification (SDA) to genomic DNA extracted from single spores of Glomus and Gigaspora species to address this problem. The genome coverage of the SDA process was evaluated by PCR amplification of the beta-tubulin1 gene and part of the rDNA cluster present in AM fungi. The fidelity of SDA was evaluated further by sequencing the Glomus intraradices ITS1 variants to detect the four ITS1 variants previously identified for this fungus.

Bacillus Phages↗

PubMed1996

Human immunodeficiency virus type 1 reverse transcriptase: role of Tyr115 in deoxynucleotide binding and misinsertion fidelity of DNA synthesis.

Tyr115 is located in the vicinity of the polymerase catalytic site of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. Site-directed mutagenesis was used to generate variant enzymes having Phe, Trp, Ala, Ser, Asp or Lys instead of Tyr115. The substitution of Tyr115 by Phe renders a fully active polymerase, displaying similar kinetic parameters, processivity and misinsertion fidelity of DNA synthesis as the wild-type enzyme. In contrast, the replacement of Tyr by Asp or Lys produced enzymes with a very low polymerase activity. The activity of the variant enzymes having Trp, Ala or Ser instead of Tyr115 was reduced significantly, particularly when poly(rA)484 was used as template. This effect was caused by a dramatic increase in the Km value for dTTP, and was detected using a DNA template mimicking a proviral HIV-1 gag sequence. Misinsertion fidelity assays revealed that mutants Y115W, Y115A and Y115S had a higher misinsertion efficiency than the wild-type reverse transcriptase. The low fidelity of these mutants appears to be related to nucleotide recognition rather than altered DNA-DNA template-primer interactions. The effects observed on the steady state kinetic constants, processivity and fidelity were mediated by the 66 kDa subunit, as demonstrated using chimeric heterodimers with the Y115A substitution in either p66 or p51.

DNA Replication↗

PubMed1998

Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase.

For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.

DNA-Directed DNA Polymerase↗

PubMed1980

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta.

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.

Animals↗

PubMedDate not supplied

[Molecular mechanisms of spontaneous transversions and transitions].

Molecular mechanisms of spontaneous base substitutions during template directed nucleic acid synthesis have been considered. For this purpose calculations of interaction energy in various coplanar base pairs with approximately such glycoside bond positions as for Watson--Crick base pairs have been carried out by means of atom-atom potential function method. A number of this energy local minima which could be realized in double helix during template synthesis have been found. The calculation results permit to present purine-purine pair formation with transition of one of nucleotides to syn-conformation as the main transversion pathway and formation of wobble pairs UG and AC as the transition pathway. A possible contribution of interactions between base pairs and enzyme to fidelity of template synthesis have been evaluated. Calculation results are in accord with experimental data on spontaneous mutation frequencies of errors in nucleic acid synthesis in vitro.

DNA↗

PubMed1998

Virtues of being faithful: can we limit the genetic variation in human immunodeficiency virus?

Human immunodeficiency virus (HIV) infections are characterized by a high degree of viral variation. The genetic variation is thought to be a combined effect of a high error rate of reverse transcriptase (RT), viral genomic recombination, the selection forces of the human immune system, the requirement for growth in multiple cell types during pathogenesis, and persistent immune activation associated with HIV disease. This hypermutability gives the virus an ability to escape mechanisms of innate immune surveillance and therapeutic interventions. Indeed, HIV variants that are resistant to drugs that antagonize both the HIV protease and RT enzymes are well described. Furthermore, there are seemingly no procedures to restrict this disarming property of HIV to mutate rapidly. Recently we have shown that some of the drug-resistant RTs display an increased in vitro polymerase fidelity. The question is whether this finding will stimulate new approaches that will not only help the immune system to deal with the virus more efficiently but also to reduce or delay resistance to various classes of anti-HIV drugs. The pros and cons of this concept and the influence of viral replication rates and viral fitness on HIV variability are discussed.

Drug Resistance, Microbial↗

PubMed2004

High fidelity of yellow fever virus RNA polymerase.

Three consecutive plaque purifications of four chimeric yellow fever virus-dengue virus (ChimeriVax-DEN) vaccine candidates against dengue virus types 1 to 4 were performed. The genome of each candidate was sequenced by the consensus approach after plaque purification and additional passages in cell culture. Our data suggest that the nucleotide sequence error rate for SP6 RNA polymerase used in the in vitro transcription step to initiate virus replication was as high as 1.34 x 10(-4) per copied nucleotide and that the error rate of the yellow fever virus RNA polymerase employed by the chimeras for genome replication in infected cells was as low as 1.9 x 10(-7) to 2.3 x 10(-7). Clustering of beneficial mutations that accumulated after multiple virus passages suggests that the N-terminal part of the prM protein, a specific site in the middle of the E protein, and the NS4B protein may be essential for nucleocapsid-envelope interaction during flavivirus assembly.

DNA-Directed RNA Polymerases↗

PubMed1999

A mutation detected in DNA polymerase delta cDNA from Novikoff hepatoma cells correlates with abnormal catalytic properties of the enzyme.

Tumor development is characterized by accumulation of mutations. Such mutations, if induced by carcinogens in DNA polymerase genes, would confer mutator properties on the DNA replication machinery, even at later stages of development. To investigate whether DNA polymerase delta can be mutated, we compared these enzymes from highly malignant Novikoff hepatoma cells and from regenerating normal rat liver. We sequenced the DNA polymerase delta cDNA from both sources and investigated the physico-chemical properties, inhibition characteristics, and copying fidelity of the purified enzymes. The cDNA sequences examined included the entire reading frame encoding the catalytic subunit (subunit I) of DNA polymerase delta. First-strand cDNAs were prepared from total RNA of both normal rat liver and Novikoff cells by reverse transcription, and the polymerase delta sequences were amplified by the polymerase chain reaction. cDNA (3325 bp) were sequenced. A single heterozygous mutation (CGG --> CAG) has been detected in nucleotide position 1948 (codon 648) of the polymerase delta gene from Novikoff cells, resulting in an Arg to Gln change. Position 648 lies just proximal to the conserved region VI, which is part of the "fingers" subdomain of alpha-like polymerases. This subdomain is involved in dNTP binding. Upon comparison of biochemical characteristics of partially purified DNA polymerase delta from both Novikoff cells and rat liver, the following properties of the enzyme from Novikoff cells were found to be altered: (i) K(50) values for nucleotide analogs (e.g. butylphenyl-dGTP) were lower, (ii) sensitivity to various antineoplastic drugs (e.g. doxorubicin, topotecan and distamycin) was enhanced, (iii) copying fidelity was decreased when primer templates containing O(6)-methylguanine were used, and (iv) the activity of DNA polymerase delta from Novikoff tumor cells was less stimulated by lactate dehydrogenase than the enzyme from normal cells. The altered biochemical characteristics of DNA polymerase delta from Novikoff cells suggest mutator properties. We conclude that the point mutation detected in the cDNA might be causally related to the observed changes in inhibition characteristics and copying fidelity.

Amino Acid Sequence↗

PubMed2000

Strengthening HIV prevention: application of a research-to-practice framework.

As the HIV epidemic continues to affect at-risk and vulnerable populations, providers strive to improve prevention programs, in part by seeking new interventions with greater effects. Although interventions with scientific evidence of effectiveness are vital to this effort, many challenges limit access to research products. We examine key challenges and offer a framework for moving research to practice, one in which research steps are linked to practice steps and all these activities take place in a complex and dynamic environment. The Replicating Effective Programs (REP) project of the Centers for Disease Control and Prevention and other technology transfer activities illustrate the operation of this framework for HIV prevention. Further actions to improve technology transfer are called for. These include reducing time from study design to practice; learning from field-based implementations; providing guidance about fidelity to, and tailoring of, science-based interventions; improving linkages among consumers, providers, and researchers; and seeking additional resources.

Centers for Disease Control and Prevention, U.S.↗