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At least 1,063 records · Page 59Linked to original sources
PubMed2004

Evaluating the contributions of desolvation and base-stacking during translesion DNA synthesis.

DNA polymerases catalyze the insertion of a nucleoside triphosphate into the growing polymer chain using the template strand as a guide. Numerous factors such as hydrogen bonding interactions, base-stacking contributions, and desolvation play important roles in controlling the efficiency and fidelity of this process. We previously demonstrated that 5-nitro-indolyl-2'-deoxyriboside triphosphate, a non-natural nucleobase with enhanced base-stacking properties, was more efficiently inserted opposite a non-templating DNA lesion compared to natural templating nucleobases (E. Z. Reineks and A. J. Berdis, Biochemistry, 2004, 43, 393-404). The catalytic enhancement was proposed to reflect increased base-stacking interactions of the non-natural nucleobase with the polymerase and DNA. However, the effects of desolvation could not be unambiguously refuted. To further address the contributions of base stacking and desolvation during translesion DNA replication, we synthesized indolyl-2'-deoxyriboside triphosphate, a nucleobase devoid of nitro groups, and measured its efficiency of enzymatic insertion into modified and unmodified DNA. Removal of the nitro group reduces the catalytic efficiency for insertion opposite an abasic site by 3600-fold. This results from a large decrease in the rate of polymerization (similar 450-fold) coupled with a modest decrease in binding affinity (similar 8-fold). Since both non-natural nucleobases show the same degree of hydrophobicity, we attribute this reduction to the loss of base-stacking contributions rather than desolvation capabilities. Indolyl-2'-deoxyriboside triphosphate can also be inserted opposite natural nucleobases. Surprisingly, the catalytic efficiency for insertion is nearly identical to that measured for insertion opposite an abasic site. These data are discussed within the context of pi-electron interactions of the incoming nucleobase with the polymerase:DNA complex. Despite this lack of insertion selectivity, the polymerase is unable to extend beyond the non-natural nucleobase. This result indicates that indolyl-2'-deoxyriboside triphosphate acts as an indiscriminate chain terminator of DNA synthesis that may have unique therapeutic applications.

Bacteriophages↗
PubMed2003

Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design.

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV plays a pivotal role in viral replication and is a potential target for anti-SARS therapy. There is a lack of structural or biochemical data on any coronavirus polymerase. To provide insights into the structure and function of SARS-CoV RdRp, we have located its conserved motifs that are shared by all RdRps, and built a three-dimensional model of the catalytic domain. The structural model permits us to discuss the potential functional roles of the conserved motifs and residues in replication and their potential interactions with inhibitors of related enzymes. We predict important structural attributes of potential anti-SARS-CoV RdRp nucleotide analog inhibitors: hydrogen-bonding capability for the 2' and 3' groups of the sugar ring and C3' endo sugar puckering, and the absence of a hydrophobic binding pocket for non-nucleoside analog inhibitors similar to those observed in hepatitis C virus RdRp and human immunodeficiency virus type 1 reverse transcriptase. We propose that the clinically observed resistance of SARS to ribavirin is probably due to perturbation of the conserved motif A that controls rNTP binding and fidelity of polymerization. Our results suggest that designing anti-SARS therapies can benefit from successful experiences in design of other antiviral drugs. This work should also provide guidance for future biochemical experiments.

Amino Acid Motifs↗
PubMed1983

Construction of a cloned library of adenovirus DNA fragments in bacteriophage M13.

The construction of recombinant M13 phages containing adenovirus DNA inserts was undertaken to provide strand-specific hybridization probes for analyses of adenovirus type 2 RNA transcripts. A library of molecular probes was constructed by cloning restriction endonuclease fragments of adenovirus types 2 and 5 DNA in the duplex replicative form DNA of the single-stranded bacteriophage vectors, M13mp7, M13mp8, and M13mp9 (Messing, J., and Vieira, J. (1982) Gene 19,269-276). Adenovirus DNA segments from early, intermediate, and late gene regions, accounting for at least 95% of the adenovirus chromosome, have been cloned in both possible orientations using these M13 derivatives as vectors. DNA cloned into these vectors can readily be obtained in a circular single-stranded form directly from mature phage particles. The cloned DNA fragments have been oriented and further characterized by restriction endonuclease mapping and hybridization with 32P-labeled adenovirus DNA. The polarity and fidelity of the adenovirus DNA in the recombinant phages has been confirmed by hybridization with labeled adenovirus 2 early and late mRNA. Restriction endonuclease analyses of M13 clones containing adenovirus DNA inserts spanning genome coordinates 31.7-56.9 have indicated that the relative locations of some restriction coordinates located within this region do not correspond to the mapped restriction sites in the DNA of adenovirus 2. Potential uses for these M13 clones in studies of adenovirus gene expression are discussed.

Adenoviridae↗
PubMed1998

Lamivudine resistance of HIV type 1 does not delay development of resistance to nonnucleoside HIV type 1-specific reverse transcriptase inhibitors as compared with wild-type HIV type 1.

We compared the development of resistance toward BI-RG-587 (nevirapine) and alpha-APA R89439 (loviride) starting from the wild-type HIV-1 strain IIIB and the 3TC-resistant HIV-1 strain containing the M184V mutation. The reverse transcriptase of the M184V mutant has been reported to have a higher fidelity. Our experiments showed that there was no significant delay in virus breakthrough of the M184V mutant as compared with the wild-type virus. We therefore conclude that the reported higher fidelity of the M184V mutant does not lead to a delay in the development of resistance to the nonnucleoside reverse transcriptase inhibitors nevirapine and loviride.

Acetamides↗
PubMed2006

A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus.

In (+) RNA coronaviruses, replication and transcription of the giant approximately 30 kb genome to produce genome- and subgenome-size RNAs of both polarities are mediated by a cognate membrane-bound enzymatic complex. Its RNA-dependent RNA polymerase (RdRp) activity appears to be supplied by non-structural protein 12 (nsp12) that includes an RdRp domain conserved in all RNA viruses. Using SARS coronavirus, we now show that coronaviruses uniquely encode a second RdRp residing in nsp8. This protein strongly prefers the internal 5'-(G/U)CC-3' trinucleotides on RNA templates to initiate the synthesis of complementary oligonucleotides of <6 residues in a reaction whose fidelity is relatively low. Distant structural homology between the C-terminal domain of nsp8 and the catalytic palm subdomain of RdRps of RNA viruses suggests a common origin of the two coronavirus RdRps, which however may have evolved different sets of catalytic residues. A parallel between the nsp8 RdRp and cellular DNA-dependent RNA primases is drawn to propose that the nsp8 RdRp produces primers utilized by the primer-dependent nsp12 RdRp.

Amino Acid Sequence↗
PubMed1997

Mutational spectrometry without phenotypic selection: human mitochondrial DNA.

By first separating mutant from nonmutant DNA sequences on the basis of their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that allows observation of point mutations in biological samples at fractions at or above 10-6. Using this method, we have observed the hotspot point mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues. To date, 19 mutants have been isolated, their fractions ranging from 4x10-4 down to the limit of detection. We performed specific tests to determine if the observed signals were artefacts arising from contamination, polymerase errors during PCR or DNA adducts created during the procedure. We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, were included with true mutants in our separation steps and converted to mutants during PCR. We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at least in part from mismatch intermediates or cellular DNA adducts. This technology is therefore sufficient for the observation of the spectrum of point mutations in human mitochondrial DNA and is a tool for discovering the primary causes of these mutations.

Artifacts↗
PubMed1988

Fidelity of HIV-1 reverse transcriptase.

The human immunodeficiency virus type 1 (HIV-1) shows extensive genetic variation and undergoes rapid evolution. The fidelity of purified HIV-1 reverse transcriptase was measured during DNA polymerization in vitro by means of three different assays. Reverse transcriptase from HIV-1 introduced base-substitution errors in DNA from the bacteriophage phi X174 amber3 at estimated frequencies of 1/2000 to 1/4000. Analyses of misincorporation rates opposite a single template adenine residue showed that HIV-1 reverse transcriptase catalyzed nucleotide mismatches with a specificity of A:C much greater than A:G greater than A:A. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately five to ten errors per HIV-1 genome per round of replication in vivo. This high error rate suggests that misincorporation by HIV-1 reverse transcriptase is, at least in part, responsible for the hypermutability of the AIDS virus. The specificity of misincorporation may provide a basis for the systematic construction of antiviral nucleosides.

Avian Myeloblastosis Virus↗
PubMed2003

Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli.

Cells exposed to DNA damaging agents in their natural environment do not undergo continuous cycles of replication but are more frequently engaged in gene transcription. Luciferase gene expression analysis with DNA templates containing uracil or 8-oxoguanine, placed at a defined position, indicated that in nondividing Escherichia coli cells, efficient mutagenic lesion bypass does occur in vivo during transcription. Sequence analyses of the transcript population revealed that RNA polymerase inserts adenine opposite to uracil, and adenine or cytosine opposite to 8-oxoguanine. Surprisingly, deletions were also detected for 8-oxoguanine-containing templates, indicating RNA polymerase slippage over this lesion. Genetic analyses showed that, in E. coli, 8-oxoguanine is subject to transcription-coupled repair. Consequently, DNA damages alter transcription fidelity in vivo, which may lead to the production of mutant proteins that have the potential to change the phenotype of nondividing cells.

Base Sequence↗
PubMed1989

Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.

Base Sequence↗
PubMed2002

TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing.

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.

Bacillus Phages↗
PubMed2003

Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis.

We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5'-->3' direction; the appropriate template for any gene expression analysis.

Cell Line↗
PubMed1992

Mutagenesis by O6 meG residues within codon 12 of the human Ha-ras proto-oncogene in monkey cells.

The first or/and the second guanines of the human Ha-ras codon 12 (normally GGC) were substituted by O6 meG residues and the modified sequence was subsequently introduced into an SV40-based shuttle vector able to replicate in both simian cells and bacteria. After replication in simian COS7 cells (proficient in O6-alkyl-guanine transferase), plasmid DNA was extracted and mutations were screened in E. coli DH5 alpha cells. The vast majority of the mutations induced by O6 meG were G----A transitions. The mutation frequency observed at the second guanine of codon 12 (12G2 position: 3.75% +/- 0.4) was higher than the one observed at the first guanine (12G1 position: 1.09% +/- 0.6). This difference was confirmed by the results obtained when two adjacent O6 meG residues were positioned within codon 12. The higher mutation frequency observed for the 12G2 position could be attributed to differential repair or/and variation in polymerase fidelity. These results are in agreement with animal experiments where alkylating agents gave rise to mutation on G2 position of codon 12.

Animals↗
PubMed2000

Intranuclear trafficking of transcription factors: implications for biological control.

The subnuclear organization of nucleic acids and cognate regulatory factors suggests that there are functional interrelationships between nuclear structure and gene expression. Nuclear proteins that are localized in discrete domains within the nucleus include the leukemia-associated acute myelogenous leukemia (AML) and promyelocytic leukemia (PML) factors, the SC-35 RNA-processing factors, nucleolar proteins and components of both transcriptional and DNA replication complexes. Mechanisms that control the spatial distribution of transcription factors within the three-dimensional context of the nucleus may involve the sorting of regulatory information, as well as contribute to the assembly and activity of sites that support gene expression. Molecular, cellular, genetic and biochemical approaches have identified distinct protein segments, termed intranuclear-targeting signals, that are responsible for directing regulatory factors to specific subnuclear sites. Gene rearrangements that remove or alter intranuclear-targeting signals are prevalent in leukemias and have been linked to altered localization of regulatory factors within the nucleus. These modifications in the intranuclear targeting of transcription factors might abrogate fidelity of gene expression in tumor cells by influencing the spatial organization and/or assembly of machineries involved in the synthesis and processing of gene transcripts.

Biological Transport↗
PubMed2003

Centrosome abnormalities and genomic instability induced by human papillomavirus oncoproteins.

Cervical cancer is tightly associated with infection by high-risk human papillomaviruses (HPVs). Many high-risk HPV-positive lesions are genomically unstable and show chromosomal gains and losses already at early stages of carcinogenic progression. These genomic aberrations are caused by the HPV-encoded oncoproteins E6 and E7, which subvert mitotic fidelity of the infected host cell. Whereas E7 drives genomic instability by inducing abnormal centrosome numbers, E6 cooperates with E7 presumably by relaxing critical checkpoint control mechanisms. The ability of E7 to induce centrosome duplication errors (CDEs) may be linked to the re-programming of the host cell cycle machinery, including dysregulation of cyclin/cyclin-dependent kinase (cdk) 2 activity. Given the role of cdk2 as a regulatory node not only for cell cycle progression but also for centrosome duplication, inhibition of cdk2 may not only retard cellular proliferation but also decrease CDEs and centrosome-related mitotic defects. Compared to some conventional cytotoxic agents, which exclusively target DNA replication, modulation of cdk2 activity may hold the promise of diminishing the development of genomically unstable, aneuploid tumor cells that are frequently the source of chemotherapy resistance in malignant tumors.

Animals↗
PubMed2005

Retrovirus reverse transcriptases containing a modified YXDD motif.

The YXDD motif, where X is a variable amino acid, is highly conserved among various viral RNA-dependent DNA polymerases. Mutations in the YXDD motif can abolish enzymatic activity, alter the processivity and fidelity of enzymes and decrease virus infectivity. This review provides a summary of the significant documented studies on the YXDD motif of HIV-1, simian immunodeficiency virus, feline immunodeficiency virus and murine leukaemia virus and the impact of mutation that this motif has had on viral pathogenesis and drug treatment.

Amino Acid Motifs↗
PubMed2001

Control of branch-site choice by a group II intron.

The branch site of group II introns is typically a bulged adenosine near the 3'-end of intron domain 6. The branch site is chosen with extraordinarily high fidelity, even when the adenosine is mutated to other bases or if the typically bulged adenosine is paired. Given these facts, it has been difficult to discern the mechanism by which the proper branch site is chosen. In order to dissect the determinants for branch-point recognition, new mutations were introduced in the vicinity of the branch site and surrounding domains. Single mutations did not alter the high fidelity for proper branch-site selection. However, several combinations of mutations moved the branch site systematically to new positions along the domain 6 stem. Analysis of those mutants, together with a new alignment of domain 5 and domain 6 sequences, reveals a set of structural determinants that appear to govern branch-site selection by group II introns.

Base Sequence↗
PubMed1979

Conservation of repeated DNA sequences in aneuploid human tumor cells.

A series of human neuroectodermal tumors, all containing more than the normal diploid DNA, and each with its own distinct chromosome mode, were studied using restriction enzyme cleavage and specific DNA sequence hybridization. Methods described were quite sensitive and quantitative and as few as 40 molecules with a given restriction site were reproducibly detected in total nuclear DNA. Analysis of several fluorescent gel bands associated with different chromosomal domains revealed no changes between any of the tumor and normal cells. Specific probe hybridization, using purified complex repeating sequences, indicated fidelity of base sequence, as well as preservation of the relative amounts of each of a number of minor related multimers in both the tumor and normal cells. Centromeric regions containing arrays of such sequences may be maintained in these tumor cells and furthermore it is possible that some of these cells are polyploid with respect to DNA sequences, rather than aneuploid as their chromosome profiles suggest.

Aneuploidy↗
PubMed1999

Variability in spine loading model performance.

OBJECTIVE: To assess the sources of variability associated with an EMG-assisted model of spine loading. DESIGN: In vivo measurements of trunk dynamics, lifting moments and muscle activities were used as inputs into an EMG-assisted spine loading model. BACKGROUND: Several types of variability are inherent in biomechanical assessments of risk associated with trunk bending motions during lifting. Variability may occur as a function of variations in spine loading due to either subject variations in motion profiles (kinematics) or biomechanical model performance. METHODS: Twelve experienced and inexperienced materials handlers performed 10 repeated lifts where load weight, asymmetry, and velocity were varied. The experiment was replicated on a second day to assess day to day variability. RESULTS: These model performance variables indicated that variability was mainly a function of subject characteristics and experience. Minor variations in variability were associated with the task asymmetry and weight lifted. Advanced analyses suggested that experienced workers had a greater range of back motion compared to inexperienced workers which would affect the length-strength component of the model calibration. CONCLUSIONS: This study indicates that for the results of an EMG-assisted model to be accurate, it is important to ensure that the model reflects a realistic relationship between the trunk muscle length and the muscle force production capacity. Underestimation if this relationship can degrade model fidelity and robustness. RELEVANCE: These results imply that by properly calibrating the model it is then reasonable to assume that the vast majority of variations observed in repeated exertions of a particular trial are due to kinematic and kinetic differences inherent in the muscle control system and not a function of model randomness.

Adult↗