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At least 991 records · Page 55Linked to original sources
PubMed1984

Fidelity of transcription of Xenopus laevis globin genes injected into Xenopus laevis oocytes and unfertilized eggs.

The Xenopus laevis alpha 1- and beta 1-globin genes were injected into oocytes and unfertilized eggs of X. laevis. In oocytes, the injected globin genes were actively transcribed, but the majority of the transcripts were incorrectly initiated. In unfertilized eggs, the injected genes were transcribed at a low level but only from the correct start sites. In oocytes, the injected circular plasmid DNA containing the cloned globin genes persisted but did not replicate. In contrast, DNA injected into unfertilized eggs replicated up to 15-fold within a 22-h period. We suggest that the ability of the egg to selectively transcribe the injected X. laevis globin genes from the correct promoter sites may be related to differences in chromatin structure between the oocyte and the unfertilized egg.

Amanitins↗
PubMed2002

A reexamination of the nucleotide incorporation fidelity of DNA polymerases.

Intensive study has been devoted to understanding the kinetic and structural bases underlying the exceptionally high fidelity (low error frequencies) of the typical DNA polymerase. Commonly proposed explanations have included (i) the concept of fidelity check points, in which the correctness of a nascent base pair match is tested at multiple points along the reaction pathway, and (ii) an induced-fit fidelity enhancement mechanism based on a rate-limiting, substrate-induced conformational change. In this article, we consider the evidence and theoretical framework for the involvement of such mechanisms in fidelity enhancement. We suggest that a "simplified" model, in which fidelity is derived fundamentally from differential substrate binding at the transition state of a rate-limiting chemical step, is consistent with known data and sufficient to explain the substrate selectivity of these enzymes.

DNA Replication↗
PubMed2003

Structural insights into the origins of DNA polymerase fidelity.

DNA polymerases discriminate from a pool of structurally similar molecules to insert the correct nucleotide to preserve Watson-Crick base pairing rules. The ability to choose between "right and wrong" is highly dependent on the identity of the polymerase. Because naturally occurring polymerases with divergent fidelities insert incorrect nucleotides with comparable efficiencies, fidelity is primarily governed by the ability to insert the correct nucleotide. DNA polymerases generally bind the correct nucleotide with similar affinities, but low-fidelity polymerases insert correct nucleotides more slowly than higher fidelity enzymes. A comparison of crystallographic ternary substrate complexes of DNA polymerases from five families exhibiting a range of nucleotide insertion rates reveals possible structural features that lead to rapid, efficient, and faithful DNA synthesis.

Animals↗
PubMed1979

Single-strand binding protein enhances fidelity of DNA synthesis in vitro.

The effect of Escherichia coli single-strand binding protein on the accuracy of in vitro DNA synthesis has been determined by using two independent methods. By using the synthetic polynucleotide poly[d(A-T)] and measuring dGTP misincorporation or by using phi X174 DNA and measuring nucleotide substitutions, we found that binding protein increases the fidelity of DNA synthesis by as much as 10-fold. This increase is observed with DNA polymerases of divergent sources and is progressive with increasing concentration of binding protein. The increased accuracy observed with DNA polymerases lacking a 3' leads to 5' exonuclease points to a mechanism other than augmented proofreading. In accord with the properties of single-strand binding proteins, it is suggested that increased fidelity is a result of enhanced base selection by the DNA polymerase, resulting from increased rigidity of the template due to its interaction with binding protein.

Base Sequence↗
PubMed1998

The upf3 protein is a component of the surveillance complex that monitors both translation and mRNA turnover and affects viral propagation.

The nonsense-mediated mRNA decay pathway functions to degrade aberrant mRNAs that contain premature translation termination codons. In Saccharomyces cerevisiae, the Upf1, Upf2, and Upf3 proteins have been identified as trans-acting factors involved in this pathway. Recent results have demonstrated that the Upf proteins may also be involved in maintaining the fidelity of several aspects of the translation process. Certain mutations in the UPF1 gene have been shown to affect the efficiency of translation termination at nonsense codons and/or the process of programmed -1 ribosomal frameshifting used by viruses to control their gene expression. Alteration of programmed frameshift efficiencies can affect virus assembly leading to reduced viral titers or elimination of the virus. Here we present evidence that the Upf3 protein also functions to regulate programmed -1 frameshift efficiency. A upf3-Delta strain demonstrates increased sensitivity to the antibiotic paromomycin and increased programmed -1 ribosomal frameshift efficiency resulting in loss of the M1 virus. Based on these observations, we hypothesize that the Upf proteins are part of a surveillance complex that functions to monitor translational fidelity and mRNA turnover.

Alleles↗
PubMed1981

Metal-induced infidelity of DNA synthesis.

In this paper, we consider the effects of metal ions on the accuracy of catalysis by DNA polymerases. Certain activating and nonactivating metal ions have been shown to diminish the fidelity of DNA synthesis in vitro with a variety of DNA polymerases. There is a significant correlation between the metals that decrease fidelity and those that have been reported to be mutagenic and carcinogenic. Thus, metal carcinogens are no exception to the general postulate that carcinogens can be identified by their interactions with DNA.

Animals↗
PubMed1989

Exonucleolytic proofreading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase.

DNA polymerase-primase complex, isolated with an apparently undegraded alpha-subunit, was immunoaffinity-purified to near homogeneity from the human lymphoblast line HSC93. The undegraded state of the alpha-subunit was monitored by Western-blot analysis of crude cellular extracts and all active fractions obtained during purification. The human polymerase-primase consists of four subunits with molecular weights of 195, 68, 55 and 48 kd. The fidelity of the polymerase-primase in copying bacteriophage phi X174am16 DNA in vitro was determined by measuring the frequency of production of different revertent phages. The overall accuracy was between 4 x 10(-6) and 10 x 10(-6). This value reflects the spontaneous mutation frequency of phi X174am16 phages in Escherichia coli, and is 10- to 20-fold higher than the accuracy of a conventionally purified enzyme from calf thymus. The frequencies of base pairing mismatches, estimated from pool bias measurements, were 3.5 x 10(-7) (1/2 880,000) for dGMP:Ttemplate mispairs, between 10(-7) and 10(-8) for dCMP:Ttemplate (1/35,000,000), dCMP:Atemplate (1/18,200,000) and dAMP:Gtemplate mispairs (1/16,500,000), and below 10(-8) (1/100,000,000) for dTMP:Ttemplate, dGMP:Atemplate and dGMP:Gtemplate mispairs. In contrast to previous preparations, the intact polymerase-primase possesses a 3'----5' exonuclease activity. This exonuclease removes both matched and mismatched 3'-OH ends, with a preference for mismatched bases. Fidelity was reduced 8-fold by increasing the concentration of the next nucleotide following the incorporated mismatch nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage phi X 174↗
PubMed1999

New human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) mutants with increased fidelity of DNA synthesis. Accuracy, template binding, and processivity.

Infidelity of DNA synthesis by human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) is a presumptive determinant of HIV-1 hypervariability and is incompletely understood at the mechanistic and structural levels. Amino acid substitution at only three residues, including Asp-76 (Kim, B., Hathaway, T. R., and Loeb, L. A. (1996) Biochemistry 37, 5831-5839), is known to increase fidelity. We report here that substitution at Arg-78 can also increase accuracy. Mutant R78A RT showed reduced primer extension in misincorporation assays lacking a complementary dNTP and exhibited a 9-fold decrease in mutation frequency in the M13mp2 lacZ forward mutation assay. Previous structural studies indicate that Arg-78 and Asp-76 lie in a region that interacts with template nucleotides. Interestingly, R78A RT exhibited 6- to 8-fold decreases in binding affinity (K(d)) for RNA and DNA templates relative to wild type RT. In contrast, D76V RT, which also increases fidelity (Kim et al., 1996), showed a 6- to 7-fold increased affinity. The processivity of R78A RT on both RNA and DNA templates was substantially reduced relative to wild type RT, whereas the processivity of D76V RT was increased. We discuss relationships of fidelity, template binding, and processivity in these and other HIV RT mutants.

Amino Acid Substitution↗
PubMed2004

Gene structure, purification and characterization of DNA polymerase beta from Xiphophorus maculatus.

Cloning of the Xiphophorus maculatus Polbeta gene and overexpression of the recombinant Polbeta protein has been performed. The organization of the XiphPolbeta introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Polbeta with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase beta (Polbeta) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polbeta, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolbeta for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polbeta. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolbeta and human Polbeta were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polbeta. In contrast, the recombinant XiphPolbeta displayed significantly lower fidelities than human Polbeta for dNTP insertion opposite a single-nucleotide gap at 37 degrees C.

Amino Acid Sequence↗
PubMed1991

Mechanisms responsible for the limited lifespan and immortal phenotypes in cultured mammalian cells.

Normal mammalian cells have a limited lifespan in culture and hypotheses explaining cellular senescence usually fall into one of two categories. One of these postulates that random errors or damage accumulate in essential macromolecules and eventually outstrip the cell's capacity for resynthesis and repair. The second considers the changes when immortal clones are produced from normal cells and in particular the lifespans of hybrids when cells of differing growth potentials are fused. These data can be explained by postulating that the mortal phenotype is dominant and that trans-acting growth inhibitors are involved in limiting lifespan. But the results do not indicate if the inhibitors are the primary cause of senescence or a secondary effect induced by quite different initial events. We suggest that normal cells possess proof-reading mechanisms which monitor the accuracy of chromosome segregation and replication and which can induce the synthesis of growth inhibitors when they detect major errors in chromosome metabolism. It is further postulated that random damage accumulates during the growth of normal cells and eventually leads to detectable chromosome changes and the synthesis of inhibitors. Our hypothesis predicts that the emergence of immortal clones will be linked to the absence of active inhibitors and therefore to a loss in the fidelity of chromosome metabolism. Data are quoted which show that in contrast to normal cells, immortal clones have highly irregular karyotypes, amplify segments of their chromosomes, integrate exogenous DNA efficiently, maintain a constant level of 5-methylcytosine residues and have high frequencies of chromosomal aberrations. The mechanism of the proof-reading is unknown, but it may monitor changes in the patterns by which chromosome domains are attached to the nuclear matrix.

Animals↗
PubMed1999

The base substitution fidelity of HIV-1 reverse transcriptase on DNA and RNA templates probed with 8-oxo-deoxyguanosine triphosphate.

We have used 8-O-dGTP, a mutagenic nucleotide generated by oxidative metabolism, to probe the misincorporation potential of HIV-1 reverse transcriptase (RT) during DNA synthesis templated by the same nucleotide sequence as either RNA or DNA. With either template, 8-O-dGMP was misincorporated opposite template A, yielding characteristic A-->C transversions. The error rate with DNA was similar to that with RNA, suggesting that base misincorporation by the RT during first-strand and second-strand replication may contribute equally to the HIV-1 base substitution mutation rate. The rate of 8-O-dGMP misincorporation differed by more than 10-fold among the 20 adenines in the M13mp2 template where A-->C transversions can be detected. The transversion distribution was similar with the two templates, indicating that the effects of flanking nucleotides on misincorporation rates were similar. This is consistent with structural and biochemical data suggesting that HIV-1 RT binds RNA x DNA and DNA x DNA template-primers in the same orientation. The similarities in error rates and distribution further indicate that, despite differences in the structures of free RNA x DNA and DNA x DNA duplexes (e.g., minor groove dimensions), the polymerase active site that assembles upon substrate binding establishes a similar degree of nucleotide selectivity with both types of template-primers. Comparison of the RT error distribution to that observed with two Pol I family DNA polymerases and a Pol alpha family polymerase revealed common hot and cold spots for misincorporation. This suggests that the local nucleotide sequence influences the nucleotide selectivity of four polymerases in a similar manner, despite their differences in structure, biochemical properties, and functions.

Base Pair Mismatch↗
PubMed2001

Fidelity of mitotic double-strand-break repair in Saccharomyces cerevisiae: a role for SAE2/COM1.

Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.

DNA Damage↗
PubMed2002

Dynamics of telomeric DNA turnover in yeast.

Telomerase adds telomeric DNA repeats to telomeric termini using a sequence within its RNA subunit as a template. We characterized two mutations in the Kluyveromyces lactis telomerase RNA gene (TER1) template. Each initially produced normally regulated telomeres. One mutation, ter1-AA, had a cryptic defect in length regulation that was apparent only if the mutant gene was transformed into a TER1 deletion strain to permit extensive replacement of basal wild-type repeats with mutant repeats. This mutant differs from previously studied delayed elongation mutants in a number of properties. The second mutation, TER1-Bcl, which generates a BclI restriction site in newly synthesized telomeric repeats, was indistinguishable from wild type in all phenotypes assayed: cell growth, telomere length, and in vivo telomerase fidelity. TER1-Bcl cells demonstrated that the outer halves of the telomeric repeat tracts turn over within a few hundred cell divisions, while the innermost few repeats typically resisted turnover for at least 3000 cell divisions. Similarly deep but incomplete turnover was also observed in two other TER1 template mutants with highly elongated telomeres. These results indicate that most DNA turnover in functionally normal telomeres is due to gradual replicative sequence loss and additions by telomerase but that there are other processes that also contribute to turnover.

DNA, Fungal↗
PubMed1996

The interaction of the reverse transcriptase of human immunodeficiency virus type 1 with 3'-terminally mispaired DNA.

The extension of mismatched 3'-termini of DNA was implicated as a major determinant that contributes to the low fidelity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). However, HIV-1 RT exhibits variations in its comparative efficiency to extend different 3'-mismatched base pairs that can result either from the differences in the binding capacity of the enzyme to various mispaired DNAs or from differences in the rate of extension of mispairs by a DNA-bound enzyme. In the current study we have examined the interaction of HIV-1 RT with mispaired template-primer 3'-termini, using a gel retardation assay. HIV-1 RT was found to bind mismatched template-primers with purine-pyrimidine (i.e., A . C) and purine-purine (i.e., A . A and A . G) 3'-terminal mispairs to about the same extent. Hence, HIV-1 RT can be considered (in addition to its other basic features) as a 3'-mismatched DNA binding protein. The stability of the complexes formed between HIV-1 RT and the mismatched template-primers tested seems to be unaffected significantly by neighboring sequences and by the presence of the next complementary dNTP. Thus, the dissimilarities observed previously in extension frequencies in the extension of 3'-terminal mismatches are likely to be due to an inherent property of the HIV-1 RT. The fact that HIV-1 RT binds 3'-mismatch-containing template-primers suggests that unextended mismatched DNA can undergo a rebinding process followed by a 3'-mismatch extension, contributing to further understanding of the low fidelity characteristic of HIV-1 RT. It is possible, therefore, that the interaction of the RT with the DNA may constitute an additional suitable target for the development of specific anti-HIV-1 RT drugs.

Base Sequence↗
PubMed1991

Recombinant reverse transcriptase of Rous sarcoma virus: characterization of DNA polymerase and RNAase H activities.

Enzyme preparations of Rous sarcoma virus (RSV) reverse transcriptase have been isolated from a culture of E. coli HB101(pMF14). The enzyme has been purified to homogeneity and been shown to consist of two subunits, of molecular mass 97.4 and 61.3 kDa, respectively. The optimum conditions for the DNA polymerase and RNAase H activities, fidelity of DNA synthesis on a homogeneous RNA template, and the inhibitory effect of azidothymidine triphosphate have been determined. Data on the use of RSV recombinant reverse transcriptase for cDNA synthesis are given.

Avian Sarcoma Viruses↗
PubMed2006

The DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells.

It has recently been suggested that the Saccharomyces cerevisiae protein Chl1p plays a role in cohesion establishment. Here, we show that the human ATP-dependent DNA helicase ChlR1 is required for sister chromatid cohesion in mammalian cells. Localization studies show that ChlR1 diffusely coats mitotic chromatin in prophase and then translocates from the chromatids to concentrate at the spindle poles during the transition to metaphase. Depletion of ChlR1 protein by RNA interference results in mitotic failure with replicated chromosomes failing to segregate after a pro-metaphase arrest. We show that depletion also results in abnormal sister chromatid cohesion, determined by increased separation of chromatid pairs at the centromere. Furthermore, biochemical studies show that ChlR1 is in complex with cohesin factors Scc1, Smc1 and Smc3. We conclude that human ChlR1 is required for sister chromatid cohesion and, hence, normal mitotic progression. These functions are important to maintain genetic fidelity.

Animals↗
PubMed1983

Effect of N-methyl-N'-nitro-N-nitrosoguanidine on DNA polymerases alpha and beta in vitro.

DNA polymerases alpha and beta were treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The activities of the modified DNA polymerase were decreased and the degree of lowered activity varied with the template employed. However, no decrease in fidelity was observed in DNA synthesis by the MNNG-treated polymerases. It is suggested that direct modification of the DNA polymerase molecule by MNNG is not the cause of carcinogenesis.

Animals↗
PubMed2005

A population-epigenetic model to infer site-specific methylation rates from double-stranded DNA methylation patterns.

Cytosine methylation is an epigenetic mechanism in eukaryotes that is often associated with stable transcriptional silencing, such as in X-chromosome inactivation and genomic imprinting. Aberrant methylation patterns occur in several inherited human diseases and in many cancers. To understand how methylated and unmethylated states of cytosine residues are transmitted during DNA replication, we develop a population-epigenetic model of DNA methylation dynamics. The model is informed by our observation that de novo methylation can occur on the daughter strand while leaving the opposing cytosine unmethylated, as revealed by the patterns of methylation on the two complementary strands of individual DNA molecules. Under our model, we can infer site-specific rates of both maintenance and de novo methylation, values that determine the fidelity of methylation inheritance, from double-stranded methylation data. This approach can be used for populations of cells obtained from individuals without the need for cell culture. We use our method to infer cytosine methylation rates at several sites within the promoter of the human gene FMR1.

Base Sequence↗