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PubMed2003

129-derived strains of mice are deficient in DNA polymerase iota and have normal immunoglobulin hypermutation.

Recent studies suggest that DNA polymerase eta (poleta) and DNA polymerase iota (poliota) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of poliota in generating mutations in an animal model, we first characterized the biochemical properties of murine poliota. Like its human counterpart, murine poliota is extremely error-prone when catalyzing synthesis on a variety of DNA templates in vitro. Interestingly, when filling in a 1 base-pair gap, DNA synthesis and subsequent strand displacement was greatest in the presence of both pols iota and eta. Genomic sequence analysis of Poli led to the serendipitous discovery that 129-derived strains of mice have a nonsense codon mutation in exon 2 that abrogates production of poliota. Analysis of hypermutation in variable genes from 129/SvJ (Poli-/-) and C57BL/6J (Poli+/+) mice revealed that the overall frequency and spectrum of mutation were normal in poliota-deficient mice. Thus, either poliota does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase.

Animals↗
PubMed2002

DNA polymerase III from Escherichia coli cells expressing mutA mistranslator tRNA is error-prone.

Translational stress-induced mutagenesis (TSM) refers to the elevated mutagenesis observed in Escherichia coli cells in which mistranslation has been increased as a result of mutations in tRNA genes (such as mutA) or by exposure to streptomycin. TSM does not require lexA-regulated SOS functions but is suppressed in cells defective for homologous recombination genes. Crude cell-free extracts from TSM-induced E. coli strains express an error-prone DNA polymerase. To determine whether DNA polymerase III is involved in the TSM phenotype, we first asked if the phenotype is expressed in cells defective for all four of the non-replicative DNA polymerases, namely polymerase I, II, IV, and V. By using a colony papillation assay based on the reversion of a lacZ mutant, we show that the TSM phenotype is expressed in such cells. Second, we asked if pol III from TSM-induced cells is error-prone. By purifying DNA polymerase III* from TSM-induced and control cells, and by testing its fidelity on templates bearing 3,N(4)-ethenocytosine (a mutagenic DNA lesion), as well as on undamaged DNA templates, we show here that polymerase III* purified from mutA cells is error-prone as compared with that from control cells. These findings suggest that DNA polymerase III is modified in TSM-induced cells.

Base Sequence↗
PubMed2006

The greater than twofold cost of integration for retroviruses.

Sexual reproduction, typically conceived of as a puzzling feature of eukaryotes, has posed an extraordinary evolutionary challenge in terms of the twofold replicative advantage of asexual over sexual organisms. Here we show mathematically that a greater than twofold cost is paid by retroviruses such as HIV during reverse transcription. For a retrovirus, replication is achieved through RNA reverse transcription and the effectively linear growth processes of DNA transcription during gene expression. Retroviruses are unique among viruses in that they show an alternation of generations between a diploid free living phase and a haploid integrated phase. Retroviruses engage in extensive recombination during the synthesis of the haploid DNA provirus. Whereas reverse transcription generates large amounts of sequence variation, DNA transcription is a high-fidelity process. Retroviruses come under strong selection pressures from immune systems to generate escape mutants, and reverse transcription into the haploid DNA phase serves to generate diversity followed by a phase of transcriptional clonal expansion during the restoration of diploidy from a stable, long lived, DNA encoded provirus.

Animals↗
PubMed2005

The use of thymidine analogs to improve the replication of an extra DNA base pair: a synthetic biological system.

Synthetic biology based on a six-letter genetic alphabet that includes the two non-standard nucleobases isoguanine (isoG) and isocytosine (isoC), as well as the standard A, T, G and C, is known to suffer as a consequence of a minor tautomeric form of isoguanine that pairs with thymine, and therefore leads to infidelity during repeated cycles of the PCR. Reported here is a solution to this problem. The solution replaces thymidine triphosphate by 2-thiothymidine triphosphate (2-thioTTP). Because of the bulk and hydrogen bonding properties of the thione unit in 2-thioT, 2-thioT does not mispair effectively with the minor tautomer of isoG. To test whether this might allow PCR amplification of a six-letter artificially expanded genetic information system, we examined the relative rates of misincorporation of 2-thioTTP and TTP opposite isoG using affinity electrophoresis. The concentrations of isoCTP and 2-thioTTP were optimal to best support PCR amplification using thermostable polymerases of a six-letter alphabet that includes the isoC-isoG pair. The fidelity-per-round of amplification was found to be approximately 98% in trial PCRs with this six-letter DNA alphabet. The analogous PCR employing TTP had a fidelity-per-round of only approximately 93%. Thus, the A, 2-thioT, G, C, isoC, isoG alphabet is an artificial genetic system capable of Darwinian evolution.

Base Pairing↗
PubMed1998

The mechanism of action of T7 DNA polymerase.

The recent crystal structure determination of T7 DNA polymerase complexed to a deoxynucleoside triphosphate and primer-template DNA has provided the first glimpse of a replicative DNA polymerase in a catalytic complex. The structure complements many functional and structural studies of this and other DNA polymerases, allowing a detailed evaluation of proposals for the mechanism of nucleotidyl transfer and the exploration of the basis for the high fidelity of template-directed DNA synthesis.

Binding Sites↗
PubMed1981

Deoxynucleoside [1-thio]triphosphates prevent proofreading during in vitro DNA synthesis.

The contribution of proofreading to the fidelity of catalysis by DNA polymerases has been determined with deoxyribonucleoside [1-thio]triphosphate substrates. These analogues, which contain a sulfur in place of an oxygen on the alpha phosphorus, are incorporated into DNA by DNA polymerases at rates similar to those of the corresponding unmodified deoxynucleoside triphosphates. The fidelity of DNA synthesis was measured with phi X174 am3 DNA; reversion to wild type occurs most frequently by a single base substitution, a C for a T at position 587. By using avian myeloblastosis virus DNA polymerase and DNA polymerase beta (enzymes without a proofreading 3' leads to 5' exonucleolytic activity), substitution of deoxycytidine thiotriphosphate in the reaction mixture did not alter fidelity. In contrast, with DNA polymerases from E. coli (DNA polymerase I) and bacteriophage T4 (enzymes containing a proofreading activity), fidelity was markedly reduced with deoxycytidine [1-thio]triphosphate. DNA containing phosphorothioate nucleotides is insensitive to hydrolysis by the exonuclease associated with these prokaryotic DNA polymerases. These combined results indicate that the deoxynucleoside [1-thio]triphosphates have normal base-pairing properties; however, once misinserted by a polymerase, they are not excised by proofreading. Proofreading of a C:A mismatch at position 587 is thereby found to contribute 20-fold to the fidelity of E. coli DNA polymerase I and a greater amount to the fidelity of bacteriophage T4 DNA polymerase.

Avian Myeloblastosis Virus↗
PubMed2000

Mismatch extension ability of yeast and human DNA polymerase eta.

DNA polymerase eta (Poleta) functions in error-free replication of UV-damaged DNA, and in vitro it efficiently bypasses a cis-syn T-T dimer by incorporating two adenines opposite the lesion. Steady state kinetic studies have shown that both yeast and human Poleta are low-fidelity enzymes, and they misincorporate nucleotides with a frequency of 10(-2)-10(-3) on both undamaged and T-T dimer-containing DNA templates. To better understand the role of Poleta in error-free translesion DNA synthesis, here we examine the ability of Poleta to extend from base mismatches. We find that both yeast and human Poleta extend from mismatched base pairs with a frequency of approximately 10(-3) relative to matched base pairs. In the absence of efficient extension of mismatched primer termini, the ensuing dissociation of Poleta from DNA may favor the excision of mismatched nucleotides by a proofreading exonuclease. Thus, we expect DNA synthesis by Poleta to be more accurate than that predicted from the fidelity of nucleotide incorporation alone.

Base Pair Mismatch↗
PubMed2003

Are there mutator polymerases?

DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

DNA-Directed DNA Polymerase↗
PubMed1993

Error-prone polymerization by HIV-1 reverse transcriptase. Contribution of template-primer misalignment, miscoding, and termination probability to mutational hot spots.

We have observed previously that DNA template-directed polymerization by the type 1 human immunodeficiency virus reverse transcriptase is error-prone for single-nucleotide substitution, addition and deletion errors at homopolymeric sequences. We have also noted strong termination of processive synthesis at these positions (Bebenek, K., Abbotts, J., Roberts, J. D., Wilson, S. H., and Kunkel, T. A. (1989) J. Biol. Chem. 264, 16948-16956). Here we have tested three models to explain errors at these hot spots: template-primer misalignment for deletion errors, and dislocation and direct miscoding for substitution errors. The approach involves introducing single-nucleotide changes within or flanking the homopolymeric hot spots and examining the effects that these changes have on human immunodeficiency virus type 1 (HIV-1) reverse transcriptase error rate, error specificity, and termination probability. The results obtained suggest that single-nucleotide deletion errors in homopolymeric runs result from template-primer misalignment and that both direct miscoding and template-primer dislocation contribute to the base substitution hot spots. The data also suggest that base substitution errors at one position can be templated by the preceding nucleotide or either of the next two nucleotides. Frameshift error rates at homopolymeric sites were affected by changes in the sequences flanking the runs, including single-nucleotide differences in the single-stranded template strand and in the double-stranded primer region as many as six nucleotides distant from the hot spot. Both increases and decreases in frameshift fidelity were observed, and most of these correlated with concomitant increases or decreases in the probability that HIV-1 reverse transcriptase terminated processive synthesis within the run. These data provide further support for a relationship between the frameshift fidelity and the processivity of DNA-dependent DNA synthesis by HIV-1 reverse transcriptase.

Base Sequence↗
PubMed1995

Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.

The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.

Animals↗
PubMed2000

DNA damage and repair: consequences on dose-responses.

Damage to DNA is considered to be the main initiating event by which genotoxins cause hereditary effects and cancer. Single or double strand breaks, bases modifications or deletions, intra- or interstrand DNA-DNA or DNA-protein cross-links constitute the major lesions formed in different proportions according to agents and to DNA sequence context. They can result in cell death or in mutational events which in turn may initiate malignant transformation. Normal cells are able to repair these lesions with fidelity or by introducing errors. Base excision (BER) and nucleotide excision (NER) repair are error-free processes acting on the simpler forms of DNA damage. A specialized form of BER involves the removal of mismatched DNA bases occurring as errors of DNA replication or from miscoding properties of damaged bases. Severe damage will be repaired according to several types of recombinational processes: homologous, illegitimate and site-specific recombination pathways. The loss of repair capacity as seen in a number of human genetic diseases and mutant cell lines leads to hypersensitivity to environmental agents. Repair-defective cells show qualitative (mutation spectrum) and quantitative alterations in dose-effect relationships. For such repair-deficient systems, direct measurements at low doses are possible and the extrapolation from large to low doses fits well with the linear or the linear-quadratic no-threshold models. Extensive debate still takes place as to the shape of the dose-response relationships in the region at which genetic effects are not directly detectable in repair-proficient normal cells. Comparison of repair mutants and wild-type organisms pragmatically suggests that, for many genotoxins and tissues, very low doses may have no effect at all in normal cells.

Animals↗
PubMed1995

Evolution of the cell cycle.

Cell proliferation involves duplication of all cell constituents and their more-or-less equal segregation to daughter cells. It seems probable that the performance of primitive cell-like structures would have been dogged by poor duplication and segregation fidelity, and by parasitism. This favoured evolution of the genome and with it the distinction between 'genomic' components like chromosomes whose synthesis is periodic and most other 'functional' components whose synthesis is continuous. Eukaryotic cells evolved from bacterial ancestors whose fused genome was replicated from a single origin and whose means of segregating sister chromatids depended on fixing their identity at replication. Evolution of an endo- or cytoskeleton, initially as means of consuming other bacteria, eventually enabled evolution of the mitotic spindle and a new means of segregating sister chromatids whose replication could be initiated from multiple origins. In this primitive eukaryotic cell, S and M phases might have been triggered by activation of a single cyclin-dependent kinase whose destruction along with that of other proteins would have triggered anaphase. Mitotic non-disjunction would have greatly facilitated genomic expansion, now possible due to multiple origins, and thereby accelerated the tempo of evolution when permitted by environmental conditions.

Archaea↗
PubMed1986

Trace metals and neoplasia.

Numerous trace metals induce cancerous growths in various animal species in vivo and cause mutagenic or chromosomal transformations in cells-cultured cells in vitro. The most potent is probably nickel. The present review indicates that arsenic, cadmium, chromium, nickel and probably beryllium are associated with malignant neoplasms in humans. Inhalation of these metals during processing at refineries has lead to a greater incidence of pulmonary carcinoma as well as other forms of cancer. There is an inverse relationship between the amount of selenium in the environment and the death rate from cancer in humans. Evidence is presented in this review indicating that mutagenic metal ions alter the fidelity of DNA synthesis. This has been demonstrated with purified DNA polymerases using both synthetic and natural DNA templates in vitro, and by mutagenic or carcinogenic effects in vivo. The need for further studies of the molecular effects of metal ions on DNA replication, RNA transcription and translation is indicated by these results.

Animals↗
PubMed1992

Dosage-dependent translational suppression in yeast Saccharomyces cerevisiae.

The overexpression of SUP35 (SUP2) wild-type gene, caused by increase of its copy number, induces an omnipotent suppression similar to the phenotype of mutants for this gene. The effect of extra-SUP35 was detected for moderate or even low copy number. Moreover, overdosage of the fragment including only the 5'-flanking region and N-terminal 100 bp of protein-coding sequence of SUP35 leads to allosuppression. Multi-SUP35 gene was also incompatible with extrachromosomal suppressor factor psi, presumably because of a high level of mistranslation. The suppressor effect caused by overdosage of another gene, SUP45 (SUP1), is much lower and can be detected only for one construction which is derived from high copy number plasmid. Suppression induced by extra-SUP35 and especially by extra-SUP45 is affected by the cell environment. A model predicting that the balance of gene products is a key for regulation of translational fidelity is discussed.

DNA Replication↗
PubMed1996

Lice and cospeciation: a response to Barker.

"The student who intends working on the Mallophaga should take warning that he will be tried almost beyond endurance by the paradoxes and complexities which beset his subject but he will also find, in the dual and inter-related aspect of insect and bird, an infinite fascination." (Rothschild & Clay, 1952: pp. 156-157). The study of host louse coevolution will benefit greatly from the phylogenetic perspective offered by recent advantages in molecular systematics. However, in order to make best use of phylogenies we need to appreciate the complexities of the possible relations between host and parasite phylogeny. At the same time, the very complexity of louse-host systems has a potentially useful consequence; the presence of multiple lineages of lice on the same hosts allow for replicated tests of coevolutionary hypotheses. For example, if a number of louse clades infest the same host clade but some lice show more cospeciation than others, we might ask whether there are features of louse biology that correlate with this difference in host tracking fidelity. It may further be possible to ascertain the relative importance of these features in ecological time through controlled transfer experiments. By beginning to appreciate "the paradoxes and complexities" of host-louse evolution, lice may offer us not only "infinite fascination" but also a chance to address important questions in coevolution.

Animals↗
PubMed2001

Prokaryotic DNA polymerase I: evolution, structure, and "base flipping" mechanism for nucleotide selection.

Accurate transmission of DNA material from one generation to the next is crucial for prolonged cell survival. Following the discovery of DNA polymerse I in Escherichia coli, the DNA polymerase I class of enzymes has served as the prototype for studies on structural and biochemical mechanisms of DNA replication. Recently, a series of genomic, mutagenesis and structural investigations have provided key insights into how Pol I class of enzymes function and evolve. X-ray crystal structures of at least three Pol I class of enzymes have been solved in the presence of DNA and dNTP, thus allowing a detailed description of a productive replication complex. Rapid-quench stop-flow studies have helped define individual steps during nucleotide incorporation and conformational changes that are rate limiting during catalysis. Studies in our laboratory have generated large libraries of active mutant enzymes (8000) containing a variety of substitutions within the active site, some of which exhibit altered biochemical properties. Extensive genomic information of Pol I has recently become available, as over 50 polA genes from different prokaryotic species have been sequenced. In light of these advancements, we review here the structure-function relationships of Pol I, and we highlight those interactions that are responsible for the high fidelity of DNA synthesis. We present a mechanism for "flipping" of the complementary template base to enhance interactions with the incoming nucleotide substrate during DNA synthesis.

Amino Acid Sequence↗
PubMed2001

Frequency of replication/transcription errors in (A)/(T) runs of human genes.

To estimate the error rate of the gene expression machinery and its possible age-related increase, we compared the occurrence of polymerase errors during replication and transcription in (A)/(T) runs, in DNA and RNA of young and old individuals and of early- and late-passage cultured fibroblasts. We analyzed three human genes: TPRD, TGFBR2, and ATRX containing stretches of (A)8, (A)10, and (T)13, respectively. The error rate was determined by sequencing 100 cloned PCR or RT-PCR fragments from each DNA and RNA sample. The error rates in replication and transcription increased with the stretch length. The pooled error rates for genomic DNA were: TPRD (A)8, TGFBR2 (A)10, and ATRX (T)13: 1%+/-0.41, 15.8%+/-1.3, and 31.3%+/-2.9, while those for RNA were: 3.8%+/-0.5, 19.3%+/-2.1, and 54.3%+/-1.8, respectively. The deletions of one nucleotide were the most frequent errors. In the replication analysis, a significant difference was found in old versus young individuals for the ATRX (T)13. In the transcription analysis, significantly higher error rates were obtained in old versus young individuals for the TPRD (A)8 and TGFBR2 (A)10. For these genes, the error rate in RNA isolated from fibroblasts was significantly higher than that in blood. The data show a trend of age-related increase in replication/transcription errors; however further studies are necessary to confirm this hypothesis, since the sample size is small. This imperfect fidelity of the gene expression process may explain the evolutionary disadvantage of nucleotide repeats within coding sequences, and that these repeats are targets for mutations in human diseases.

Adult↗
PubMed2003

Ribavirin causes error catastrophe during Hantaan virus replication.

Except for ribavirin, no other antiviral drugs for treating hantaviral diseases have been identified. It is well established that ribavirin will inhibit the production of infectious Hantaan virus (HTNV); however, its mechanism of action is unknown. To characterize the inhibitory effect of ribavirin on HTNV, the levels of viral RNAs, proteins, and infectious particles were measured for 3 days posttreatment of HTNV-infected Vero E6 cells. HTNV-infected cells treated with ribavirin showed a slight reduction in the levels of cRNA, viral RNA, and mRNA populations on the first day postinfection. The amount of cRNA and viral RNA increased to that observed for untreated HTNV-infected cells on day 2, whereas mRNA levels were more greatly reduced on days 2 and 3. Despite the finding of S-segment mRNA, albeit low, three of the viral proteins-nucleocapsid (N) protein and glycoproteins G1 and G2-could not be detected by immunohistochemistry in ribavirin-treated cells. To test the hypothesis that these effects were caused by incorporation of ribavirin into nascent RNA and a resultant "error catastrophe" was occurring, we cloned and sequenced the S-segment cRNA/mRNA from ribavirin-treated or untreated cells from day 3. We found a high mutation frequency (9.5/1,000 nucleotides) in viral RNA synthesized in the presence of ribavirin. Hence, the transcripts produced in the presence of the drug were not functional. These results suggest that ribavirin's mechanism of action lies in challenging the fidelity of the hantavirus polymerase, which causes error catastrophe.

Animals↗