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PubMed2002

Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli.

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.

DNA Replication↗
PubMed2001

DNA polymerase iota and related rad30-like enzymes.

Until recently, the molecular mechanisms of translesion DNA synthesis (TLS), a process whereby a damaged base is used as a template for continued replication, was poorly understood. This area of scientific research has, however, been revolutionized by the finding that proteins long implicated in TLS are, in fact, DNA polymerases. Members of this so-called UmuC/DinB/Rev1/Rad30 superfamily of polymerases have been identified in prokaryotes, eukaryotes and archaea. Biochemical studies with the highly purified polymerases reveal that some, but not all, can traverse blocking lesions in template DNA. All of them share a common feature, however, in that they exhibit low fidelity when replicating undamaged DNA. Of particular interest to us is the Rad30 subfamily of polymerases found exclusively in eukaryotes. Humans possess two Rad30 paralogs, Rad30A and Rad30B. The RAD30A gene encodes DNA polymerase eta and defects in the protein lead to the xeroderma pigmentosum variant (XP-V) phenotype in humans. Very recently RAD30B has also been shown to encode a novel DNA polymerase, designated as Pol iota. Based upon in vitro studies, it appears that Pol iota has the lowest fidelity of any eukaryotic polymerase studied to date and we speculate as to the possible cellular functions of such a remarkably error-prone DNA polymerase.

Bacterial Proteins↗
PubMed2005

Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population.

An RNA virus population does not consist of a single genotype; rather, it is an ensemble of related sequences, termed quasispecies. Quasispecies arise from rapid genomic evolution powered by the high mutation rate of RNA viral replication. Although a high mutation rate is dangerous for a virus because it results in nonviable individuals, it has been hypothesized that high mutation rates create a 'cloud' of potentially beneficial mutations at the population level, which afford the viral quasispecies a greater probability to evolve and adapt to new environments and challenges during infection. Mathematical models predict that viral quasispecies are not simply a collection of diverse mutants but a group of interactive variants, which together contribute to the characteristics of the population. According to this view, viral populations, rather than individual variants, are the target of evolutionary selection. Here we test this hypothesis by examining the consequences of limiting genomic diversity on viral populations. We find that poliovirus carrying a high-fidelity polymerase replicates at wild-type levels but generates less genomic diversity and is unable to adapt to adverse growth conditions. In infected animals, the reduced viral diversity leads to loss of neurotropism and an attenuated pathogenic phenotype. Notably, using chemical mutagenesis to expand quasispecies diversity of the high-fidelity virus before infection restores neurotropism and pathogenesis. Analysis of viruses isolated from brain provides direct evidence for complementation between members in the quasispecies, indicating that selection indeed occurs at the population level rather than on individual variants. Our study provides direct evidence for a fundamental prediction of the quasispecies theory and establishes a link between mutation rate, population dynamics and pathogenesis.

Animals↗
PubMed1980

Studies of mammalian chromosome replication. I. BrdU-induced differential staining patterns in interphase and metaphase chromosomes.

The patterns of differential staining based on the effects of BrdU-substitution in chromosomal DNA have been examined in both metaphase chromosomes and prematurely condensed chromosomes (PCC) of interphase Chinese hamster cells. Results indicate that differential staining may be obtained in chromosomes from all stages of the cell cycle and correspond to the semi-conservation mode of DNA replication. Such fidelity of differential staining in both interphase and metaphase chromosomes suggests that components essential for induction of differential staining are present throughout the cell cycle and chromosomes may contain similar structures and organization throughout the cycle.

Animals↗
PubMed2005

Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases.

DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.

DNA Primers↗
PubMed1998

Chromium(III) decreases the fidelity of human DNA polymerase beta.

Certain particulate compounds of hexavalent chromium are well-known occupational and environmental human carcinogens. Hexavalent chromium primarily enters the cells and undergoes metabolic reduction; however, the ultimate trivalent oxidation state of chromium, Cr(III), predominates within the cell. DNA-bound Cr(III) has been previously shown to decrease the fidelity of replication in the M13 phage mutation assay. This study was done to understand how Cr(III), in the presence of physiological concentrations of magnesium, affects the kinetic parameters of steady-state DNA synthesis in vitro across site-specific O6-methylguanine (m6dG) residues by DNA polymerase beta (pol beta). Cr(III) binds to the short oligomer templates in a dose-dependent manner and stimulates the activity of pol beta. Cr(III) stimulates the mutagenic incorporation of dTTP opposite m6dG more than the nonmutagenic incorporation of dCTP, and thereby Cr(III) further decreases the fidelity of DNA synthesis across m6dG by pol beta. In contrast, Cr(III) does not affect the fidelity of DNA synthesis across the normal template base, dG. Both the enhanced activity and the mutagenic lesion bypass in the presence of Cr(III) may be associated with Cr(III)-dependent stimulation of pol beta binding to DNA as reported here. This study shows some of the mechanisms by which mutagenic chromium affects DNA synthesis.

Binding Sites↗
PubMed2005

Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases.

DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity.

Amino Acid Motifs↗
PubMed2002

Checking on the fork: the DNA-replication stress-response pathway.

To ensure the fidelity of DNA replication, cells activate a stress-response pathway when DNA replication is perturbed. This pathway regulates not only progress through the cell cycle but also transcription, apoptosis, DNA repair/recombination and DNA replication itself. Mounting evidence has suggested that this pathway is important for the maintenance of genomic integrity. Here, we discuss recent findings about how this pathway is activated by replication stress and how it regulates the DNA-replication machinery to alleviate the stress.

Animals↗
PubMed1994

Genomic instability of microsatellite repeats and its association with the evolution of chronic myelogenous leukemia.

Tumorigenesis has been shown to proceed through a series of genetic alterations involving protooncogenes and tumor-suppressor genes. Investigation of genomic instability of microsatellites has indicated a new mechanism for human carcinogenesis in hereditary nonpolyposis colorectal cancer and sporadic cancer and this instability has been shown to be related to inherited predisposition to cancer. This study was conducted to determine whether such microsatellite instability is associated with the evolution of chronic myelogenous leukemia (CML) to the blast crisis. Nineteen CML patients clinically progressing from the chronic phase to accelerated phase or blast crisis and 20 other patients in the CML chronic phase were studied. By polymerase chain reaction assay, DNAs for genomic instability in five separate microsatellites in chromosome arms 5q (Mfd27), 17p (Mfd41), 18q (DCC), 3p (CI3-9), and 8p (LPL) were examined. Differences in unrelated microsatellites of chronic and blastic phase DNAs in 14 of 19 patients (73.7%) were demonstrated. Somatic instability in five microsatellites, Mfd27, Mfd41, DCC, CI3-9, and LPL, was detected in 2 of 19 (10.5%), 8 of 19 (42.1%), 11 of 19 (57.9%), 4 of 17 (23.5%), and 4 of 17 (23.5%) cases. In 10 of 19 cases (52.6%), genetic instability in at least two of five microsatellites was observed and was categorized as replication error (RER+) phenotype. CML evolution cases with myeloid, lymphoid, and mixed phenotypes and the blast crisis and accelerated phase showed somatic instability in a number of microsatellites. No alterations in leukemic cells at the chronic phase could be detected in any microsatellites. These data indicate instability of microsatellites (RER+) but not familial predisposition to possibly be a late genetic event in the evolution of CML to blast crisis. In the microsatellite of the DCC gene, complicated alterations in band patterns caused by instability as well as loss of heterozygosity (LOH) were observed in 13 of 19 cases (68.4%): instability in 9 cases, instability plus LOH in 2 cases, and only LOH in 2 cases. These highly frequent alterations in microsatellites, including instability and LOH, suggesting that secondary events due possibly to loss of fidelity in replication and repair machinery may be significantly associated with CML evolution.

Adult↗
PubMed1995

Rescue of synthetic measles virus minireplicons: measles genomic termini direct efficient expression and propagation of a reporter gene.

Measles virus (MV) mRNA transcription and replication are thought to be controlled by cis-acting sequence elements contained within the terminal MV genomic noncoding nucleotides. To validate these promoter and regulatory signal assignments, cDNAs were constructed allowing synthesis of RNAs corresponding to a MV genome in which all coding and intercistronic regions were replaced by the chloramphenicol acetyl transferase (CAT) coding sequence. Transcript production by T7 polymerase starting and ending precisely with the MV genome terminal residues was achieved by fusing the T7 polymerase promoter and the hepatitis delta virus genome ribozyme followed by tandem T7 polymerase termination sequences to the MV genomic 5' and 3' ends, respectively. Transfection of these negative polarity transcripts, mimicking natural defective interfering RNAs of the internal deletion type, into MV-infected 293 cells gave rise to CAT activity which could be serially transferred and massively amplified together with progeny helper virus in fresh cells. Transfer was blocked only by antibodies able to neutralize MV infectivity, indicating that the chimeric RNA not only was encapsidated, transcribed, and replicated, but also packaged into virions. Sequence analyses confirmed that both the expected chimeric antigenome and mRNA products were transcribed and replicated with fidelity during serial passage. Minor changes introduced in the transcription promoter markedly compromised function. This system now can be exploited to examine MV genomic cis-acting regulatory elements and extended to the development of full-length MV cDNAs.

Base Sequence↗
PubMed1992

Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4.

The DNA polymerase from the bacteriophage T4 is part of a multienzyme complex required for the synthesis of DNA. As a first step in understanding the contributions of individual proteins to the dynamic properties of the complex, e.g., turnover, processivity, and fidelity of replication, the minimal kinetic schemes for the polymerase and exonuclease activities of the gene 43 protein have been determined by pre-steady-state kinetic methods and fit by computer simulation. A DNA primer/template (13/20-mer) was used as substrate; duplexes that contained more single-strand DNA resulted in nonproductive binding of the polymerase. The reaction sequence features an ordered addition of 13/20-mer followed by dATP to the T4 enzyme (dissociation constants of 70 nM and 20 microM) followed by rapid conversion (400 s-1) of the T4.13/20-mer.dATP complex to the T4.14/20-mer.PPi product species. A slow step (2 s-1) following PPi release limits a single turnover, although this step is bypassed in multiple incorporations (13/20-mer-->17/20-mer) which occur at rates > 400 s-1. Competition between correct versus incorrect nucleotides relative to the template strand indicates that the dissociation constants for the incorrect nucleotides are at millimolar values, thus providing evidence that the T4 polymerase, like the T7 but unlike the Klenow fragment polymerases, discriminates by factors > 10(3) against misincorporation in the nucleotide binding step. The exonuclease activity of the T4 enzyme requires an activation step, i.e., T4.DNA-->T4.(DNA)*, whose rate constants reflect whether the 3'-terminus of the primer is matched or mismatched; for matched 13/20-mer the constant is 1 s-1, and for mismatched 13T/20-mer, 5 s-1. Evidence is presented from crossover experiments that this step may represent a melting of the terminus of the duplex, which is followed by rapid exonucleolytic cleavage (100s-1). In the presence of the correct dNTP, primer extension is the rate-limiting step rather than a step involving travel of the duplex between separated exonuclease and polymerase sites. Since the rate constant for 13/20-mer or 13T/20-mer dissociation from the enzyme is 6 or 8 s-1 and competes with that for activation, the exonucleolytic editing by the enzyme alone in a single pass is somewhat inefficient (5 s-1/(8 s-1+5 s-1)), ca. 40%. Consequently, a major role for the accessory proteins may be to slow the rate of enzyme.substrate dissociation, thereby increasing overall fidelity and processivity.

Base Sequence↗
PubMed1988

Fidelity of DNA synthesis in a mammalian in vitro replication system.

We have used the simian virus 40 (SV40)-based shuttle vector pZ189 in a forward-mutation assay to determine the fidelity of DNA replication in the in vitro DNA replication system developed by J.J. Li and T.J. Kelly (Proc. Natl. Acad. Sci. USA 81:6973-6977, 1984). We find that very few base substitution errors (approximately 1/180,000 bases incorporated) are made during in vitro replication of the pZ189 vector in a system derived from CV-1 monkey cells. This replication is completely dependent on added SV40 T antigen and presumably reflects synthesis that is initiated at the SV40 replication origin. The observed level of fidelity is far greater than that reported for in vitro replication of DNA by conventionally purified eucaryotic DNA polymerases alpha and beta. Thus, there must be additional cellular factors in the crude in vitro system that serve to enhance the fidelity of DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed2005

Growth, morphogenesis, and differentiation during mouse prostate development in situ, in renal grafts, and in vitro.

BACKGROUND: In vitro organ culture and renal grafting of the urogenital sinus (UGS) have both been used as models of prostate development. However, neither has been rigorously examined for its fidelity to replicate the canonical process of prostate differentiation in situ. METHODS: We assessed size, morphology, histology, and the mRNA expression of differentiation marker genes of the E14 male mouse UGS grown for 0-28 days as sub-renal capsule allografts in nude mice or in culture containing androgen and compared these to UGS development in situ. RESULTS: Development of grafted tissues was morphologically and histologically similar to development in situ but differentiation occurred more rapidly. UGS growth in organ culture resulted in bud formation, but did not trigger cellular differentiation. However, the potential for differentiation was maintained and could be rescued by grafting tissues into nude mice. CONCLUSIONS: In vitro organ culture and renal grafting of UGS tissues may be appropriate models for studying prostatic bud formation, but only grafting is an appropriate model for prostatic differentiation.

Animals↗
PubMed1997

Analysis of the mismatch and insertion/deletion binding properties of Thermus thermophilus, HB8, MutS.

The methyl-directed long patch repair pathway in Escherichia coli is involved in increasing the fidelity of replication specific repair of DNA polymerase incorporation errors. This pathway is mediated by three gene products, MutS, MutL, and MutH, which are conserved in higher eukaryotes. Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC). A MutS homologue has recently been identified in the extremely thermophilic bacterium, Thermus thermophilus. Here we describe analysis of the binding properties of this protein, which has indicated it can identify all specific base mismatches as well as one, two and three base pair insertion/deletion mutations. We therefore believe this protein may be generally useful for applications involving mismatch detection.

Adenosine Triphosphatases↗
PubMedDate not supplied

On the evolution of protein folds: are similar motifs in different protein folds the result of convergence, insertion, or relics of an ancient peptide world?

This paper presents and discusses evidence suggesting how the diversity of domain folds in existence today might have evolved from peptide ancestors. We apply a structure similarity detection method to detect instances where localized regions of different protein folds contain highly similar sequences and structures. Results of performing an all-on-all comparison of known structures are described and compared with other recently published findings. The numerous instances of local sequence and structure similarities within different protein folds, together with evidence from proteins containing sequence and structure repeats, argues in favor of the evolution of modern single polypeptide domains from ancient short peptide ancestors (antecedent domain segments (ADSs)). In this model, ancient protein structures were formed by self-assembling aggregates of short polypeptides. Subsequently, and perhaps concomitantly with the evolution of higher fidelity DNA replication and repair systems, single polypeptide domains arose from the fusion of ADSs genes. Thus modern protein domains may have a polyphyletic origin.

Amino Acid Motifs↗
PubMed1976

The effect of nitrosoguanidine upon DNA synthesis in vitro.

Both the polymerase and the exonuclease activities of DNA polymerase III are inactivated by treatment with nitrosoguanidine. The treatment of the DNA template with the mutagen does not affect the template in supporting DNA synthesis. No effect of nitrosoguanidine upon fidelity of replication in vitro was detected.

Carcinogens↗
PubMed1995

Genomic instability due to germline p53 mutations drives preneoplastic progression toward cancer in human cells.

Cells heterozygous for mutations in p53 demonstrate extreme genomic instability and develop mutations detectable at the chromosome level as well as the molecular level. This genomic instability causes initially nontumorigenic ras-expressing immortal LFS cells to progress to a tumorigenic state presumably due to additional mutational events. It is not surprising that LFS families with these p53 mutations develop the additional mutations necessary for cancer to occur at such high frequencies. This observation is consistent with increased cancer rates in these families being due to abrogation of a rate limiting step rather than a rate expected for one less step in a multistep carcinogenic process. Although p53 has been shown to be able to function as a transcription factor, mutations in p53 appear to affect genomic stability in LFS fibroblasts with double minutes and telomeric associations being prominent early events. One possibility is that p53 controls the expression of genes required for fidelity of replication or telomerase activity. Alternatively p53 may itself be a replication factor like the transcription factor CTF. In the future, we plan to investigate whether p53 plays a direct role in replication.

Animals↗
PubMed1998

Relics from the RNA world.

An RNA world is widely accepted as a probable stage in the early evolution of life. Two implications are that proteins have gradually replaced RNA as the main biological catalysts and that RNA has not taken on any major de novo catalytic function after the evolution of protein synthesis, that is, there is an essentially irreversible series of steps RNA --> RNP --> protein. This transition, as expected from a consideration of catalytic perfection, is essentially complete for reactions when the substrates are small molecules. Based on these principles we derive criteria for identifying RNAs in modern organisms that are relics from the RNA world and then examine the function and phylogenetic distribution of RNA for such remnants of the RNA world. This allows an estimate of the minimum complexity of the last ribo-organism-the stage just preceding the advent of genetically encoded protein synthesis. Despite the constraints placed on its size by a low fidelity of replication (the Eigen limit), we conclude that the genome of this organism reached a considerable level of complexity that included several RNA-processing steps. It would include a large protoribosome with many smaller RNAs involved in its assembly, pre-tRNAs and tRNA processing, an ability for recombination of RNA, some RNA editing, an ability to copy to the end of each RNA strand, and some transport functions. It is harder to recognize specific metabolic reactions that must have existed but synthetic and bio-energetic functions would be necessary. Overall, this requires that such an organism maintained a multiple copy, double-stranded linear RNA genome capable of recombination and splicing. The genome was most likely fragmented, allowing each "chromosome" to be replicated with minimum error, that is, within the Eigen limit. The model as developed serves as an outgroup to root the tree of life and is an alternative to using sequence data for inferring properties of the earliest cells.

Evolution, Molecular↗