SEARCH · Search PubMed

Results for “replication fidelity”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources

PubMed2001

Evading the proofreading machinery of a replicative DNA polymerase: induction of a mutation by an environmental carcinogen.

DNA replication fidelity is dictated by DNA polymerase enzymes and associated proteins. When the template DNA is damaged by a carcinogen, the fidelity of DNA replication is sometimes compromized, allowing mispaired bases to persist and be incorporated into the DNA, resulting in a mutation. A key question in chemical carcinogenesis by metabolically activated polycyclic aromatic hydrocarbons (PAHs) is the nature of the interactions between the carcinogen-damaged DNA and the replicating polymerase protein that permits the mutagenic misincorporation to occur. PAHs are environmental carcinogens that, upon metabolic activation, can react with DNA to form bulky covalently linked combination molecules known as carcinogen-DNA adducts. Benzo[a]pyrene (BP) is a common PAH found in a wide range of material ingested by humans, including cigarette smoke, car exhaust, broiled meats and fish, and as a contaminant in other foods. BP is metabolically activated into several highly reactive intermediates, including the highly tumorigenic (+)-anti-benzo[a]pyrene diol epoxide (BPDE). The primary product of the reaction of (+)-anti-BPDE with DNA, the (+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-dG ((+)-ta-[BP]G) adduct, is the most mutagenic BP adduct in mammalian systems and primarily causes G-to-T transversion mutations, resulting from the mismatch of adenine with BP-damaged guanine during replication. In order to elucidate the structural characteristics and interactions between the DNA polymerase and carcinogen-damaged DNA that allow a misincorporation opposite a DNA lesion, we have modeled a (+)-ta-[BP]G adduct at a primer-template junction within the replicative phage T7 DNA polymerase containing an incoming dATP, the nucleotide most commonly mismatched with the (+)-ta-[BP]G adduct during replication. A one nanosecond molecular dynamics simulation, using AMBER 5.0, has been carried out, and the resultant trajectory analyzed. The modeling and simulation have revealed that a (+)-ta-[BP]G:A mismatch can be accommodated stably in the active site so that the fidelity mechanisms of the polymerase are evaded and the polymerase accepts the incoming mutagenic base. In this structure, the modified guanine base is in the syn conformation, with the BP moiety positioned in the major groove, without interfering with the normal protein-DNA interactions required for faithful polymerase function. This structure is stabilized by a hydrogen bond between the modified guanine base and dATP partner, hydrophobic interactions between the BP moiety and the polymerase, a hydrogen bond between the modified guanine base and the polymerase, and several hydrogen bonds between the BP moiety and polymerase side-chains. Moreover, the G:A mismatch in this system closely resembles the size and shape of a normal Watson-Crick pair. These features reveal how the polymerase proofreading machinery may be evaded in the presence of a mutagenic carcinogen-damaged DNA, so that a mismatch can be accommodated readily, allowing bypass of the adduct by the replicative T7 DNA polymerase.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

How variable is a spontaneous mutation rate in cultured mammalian cells?

The Luria-Delbrück fluctuation analysis provides a method to estimate mutation rates and is commonly applied in somatic cell genetics and in cancer biology. We developed an assay for a Luria-Delbrück fluctuation analysis using the mouse lymphoma cell line, GRSL13. As these cells grow in suspension, one can handle hundreds of parallel cultures using multiwell dishes and dispensers. This assay thereby allows not only an accurate determination of the mutation rate per cell generation but also makes it possible to determine at which time after seeding mutations take place. Using approx. 8000 parallel cultures it has been possible to test whether the mutation rate is constant during the assay. It has been found that the spontaneous mutation rate of GRSL13 cells decreases in the course of a fluctuation test from 2 x 10(-6) to about 2 x 10(-7)/cell/generation. It was shown that this increased replication fidelity may partly be caused by cell density: maintenance of cells at high cell density resulted in a spontaneous mutation rate of 0.7 +/- 4.0 x 10(-7) compared to 4.0 +/- 3.1 x 10(-7) for the standard protocol. In contrast, growing the cells at extremely low cell density resulted in an enhanced mutation rate of 7.7 +/- 1.3 x 10(-7). Thus altogether the mutation rate can vary from 2 x 10(-6) to 0.7 x 10(-7) (approx. 30-fold). These results show that the spontaneous mutation rate is not constant, but highly dependent on experimental conditions. As incomplete expression and metabolic cooperation cannot explain the findings, the data suggest that the fidelity of DNA replication is not fixed but open to variation. Hence, determination of replication infidelity in cultured cells needs rigorous standardization or/and application of controlled variation in culture conditions.

Animals↗

PubMedDate not supplied

Effects of chromium(III) on DNA replication in vitro.

A number of metal compounds are important environmental carcinogens; however, the molecular mechanisms of metal-induced genotoxicity are not yet understood. Chromium, for example, is substantially mutagenic in vivo and has been shown to decrease the DNA replication fidelity in vitro. But the mechanism of chromium-induced mutagenesis is unknown and the role of replication fidelity in chromium-induced carcinogenesis is unclear. We have used in vitro DNA replication assays to investigate the effects of chromium ions on DNA polymerase activity preliminary to studying their role in chromium-induced mutagenesis. Biologically active M13mp2 DNA was replicated with purified DNA polymerases in the presence of micromolar amounts of chromium with or without the normal divalent cation, magnesium. Nucleotide incorporation kinetics were determined and sequence specific pausing was analyzed by primer-extension. Our results have demonstrated an unexpected polymerase activation by low (0.5-5.0 microns) concentrations of chromium (III), although higher concentrations of chromium are increasingly inhibitory. The increased incorporation seem at low chromium(III) concentrations is the result of increased enzyme processivity and is not polymerase specific. The possible relationship between processivity and metal-ion mutagenesis is discussed.

Chromium↗

PubMed1993

Frameshift fidelity during replication of double-stranded DNA in HeLa cell extracts.

The processes by which minus-one frameshifts arise during replication of double-stranded DNA by a human replication apparatus were examined. Using M13mp2 DNA containing the simian virus 40 (SV40) origin of replication and a plus-one frameshift mutation in the lacZ alpha reporter gene, we performed replication reactions using a HeLa cell extract and the SV40 large T antigen. Frameshifts that restore the reading frame to give a blue-plaque phenotype include the loss of one of five consecutive A.T base pairs or any one of 36 non-reiterated base pairs. Although both types of deletions were generated at rates substantially above the background mutant frequency of unreplicated DNA, the rate was highest at the A.T run, suggesting the involvement of a misaligned replication intermediate at this homopolymeric sequence. The error rate for both types of deletions increased as the concentration of dNTPs was increased. A small increase in error rate at the run of A.T base pairs was also observed when a dNMP was added to the replication reaction. These results are consistent with the correction of frameshift intermediates during replication by exonucleolytic proofreading. To examine frameshift error rates on the leading and lagging strands, we compared reversion frequencies for two vectors containing the origin of replication close to, but on opposite sides of, the mutational target. To generate strand-specific errors, nucleotide substrate imbalances were used in replication reactions with these vectors. The results suggest that there is less than a 2-fold difference in the fidelity of leading- and lagging-strand synthesis for deletions at the run of A.T base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

PubMed1985

Reduced in vivo mutagenesis by mutant herpes simplex DNA polymerase involves improved nucleotide selection.

We present evidence that mutation frequencies in a mammalian system can vary according to the replication fidelity of the DNA polymerase. We demonstrated previously that several derivatives of herpes simplex virus type 1 that encode polymerases resistant to various antiviral drugs (e.g., nucleotide analogues) also produce reduced numbers of spontaneous mutants. Here we show that the DNA polymerase from one antimutator virus exhibits enhanced replication fidelity. First, the antimutator virus showed a reduced response to known mutagens that promote base mispairing during DNA replication (N-methyl-N'-nitro-N-nitrosoguanidine, 5-bromo-deoxyuridine). Second, purified DNA polymerase from the antimutator produced fewer replication errors in vitro, based on incorporation of mispaired nucleotides or analogues with abnormal sugar rings. We have investigated possible mechanisms for the enhanced fidelity of the antimutator polymerase. We show that the mutant enzyme has altered interactions with nucleoside triphosphates, as indicated by its resistance to nucleotide analogues and elevated Km values for normal nucleoside triphosphates. We present evidence against increased proofreading by an associated 3',5' exonuclease (as seen for T4 bacteriophage antimutator polymerases), based on nuclease levels in the mutant polymerase. We propose that reduced affinity of the polymerase for nucleoside triphosphates accounts for the antimutator phenotype by accentuating differences in base-pair stability, thus facilitating selection of correct nucleotides.

Alkylation↗

PubMed1983

Products of bacteriophage T4 genes 32 and 45 improve the accuracy of DNA replication in vitro.

The six "accessory" proteins of the bacteriophage T4 specified by replication genes 32, 41, 44, 45, 61, and 62 were studied for their ability to enhance the accuracy with which phage T4 DNA polymerase (product of gene 43) replicates synthetic homopolymer duplexes in vitro. Two of these proteins, gene 32-protein (helix-destabilizing protein) and gene 45-protein, inhibited the selection of incorrect, but not correct, precursors, at the growing strand end. Gene 32-protein is shown to enhance replication fidelity by interacting with the DNA, whereas gene 45-protein exerts its fidelity-enhancing effect by interacting with the DNA polymerase. This is the first example to our knowledge of a DNA polymerase's accuracy being altered through interaction with another protein. Possible mechanisms by which gene 32- and gene 45-protein act to enhance replication fidelity are discussed.

DNA Replication↗

PubMed2001

Exonucleolytic proofreading by p53 protein.

The tumour suppressor p53 protein plays an important role in maintaining genetic integrity. Recently, p53 was shown to have an intrinsic 3'-->5' exonuclease activity. The current study has extended the characterization of purified wild-type recombinant p53-associated 3'-->5' exonuclease function to demonstrate proofreading activity. p53-associated 3'-->5' exonuclease shows clear preference for degradation of ssDNA over dsDNA substrate. On partial duplex structures, this exonucleolytic activity displays a marked preference for excision of a mismatched vs. a correctly paired 3' terminus which enables the p53 protein to act as a proofreader. However, p53 displays variation in excision of mismatched base pairs. The results demonstrate that p53 exhibits mispair excision with a specificity of A:A > A:G > A:C opposite the template adenine residue and with a specificity of G:A > G:G > G:T opposite the template guanine residue. Hence, the observed specificity of mismatch excision shows that p53 exonucleolytic proofreading preferentially repairs transversion mutations. As part of an investigation of the functional interaction between p53 and DNA polymerase, the influence of p53 on the accuracy of DNA synthesis was determined with exonuclease-deficient murine leukemia virus (MLV) reverse transcriptase (RT), representing a relatively low fidelity enzyme. Using an in vitro biochemical assay with 3'-terminal mismatch-containing DNA template primers, it was shown that wild-type recombinant p53 protein enhanced the DNA replication fidelity of MLV RT. A functional interaction between the exonuclease (p53) and polymerase (MLV RT) activities was observed; excision of mispairs by p53 was followed by further elongation onto correctly base-paired 3'-termini by MLV RT. Furthermore, the formation of 3'-mispair and subsequent mispair extension by the enzyme were decreased substantially in the presence of p53. The fact that the exonuclease-deficient MLV RT is more accurate in the presence of p53, suggests that p53 protein may function as an external proofreading exonuclease for viral enzyme. The observed decrease in initial nucleotide misincorporation and 3'-terminal mispair extension by MLV RT in the presence of p53, indicates the mechanism by which p53 affects the DNA replication fidelity of exonuclease-deficient DNA polymerase.

Baculoviridae↗

PubMed1998

Human breast cancer cells contain an error-prone DNA replication apparatus.

The mechanisms responsible for creating genetic errors and genomic instability in cancer cells have not been fully defined. Recently, it has been shown that human cells contain a highly organized complex of proteins, termed the DNA synthesome, that is fully competent to carry out all phases of SV40 in vitro DNA replication (J. M. Coll et al, Oncol. Res., 8: 435-447, 1996; L. H. Malkas et al., Biochemistry, 29: 6362-6374, 1990; Y. Wu et al., J. Cell. Biochem., 54: 32-46, 1994; N. Applegren et al., J. Cell. Biochem., 54: 32-46, 1994). DNA replication fidelity analyses of the DNA synthesome derived from malignant and nonmalignant human breast cells demonstrate that the malignant cell synthesome is mutagenic. The decrease in tumor cell replication fidelity was not due to an increased proliferative capacity of the tumor cells or an increase in the synthetic activity of their DNA synthesome. The ratios of insertions, deletions, and mismatches created by the synthesome from malignant and nonmalignant breast cells were essentially identical, despite the greater overall number of mutations made by the breast cancer cell synthesome. These data define, for the first time, a mechanism unique to cancer cells that contributes to the observed increase in genetic mutation in cancer cells.

Adult↗

PubMed2003

26th Lauriston S. Taylor Lecture: developing mechanistic data for incorporation into cancer and genetic risk assessments: old problems and new approaches.

The theme that runs through this 26th Taylor Lecture is the question of how can data on the mechanism of induction of genetic alterations by radiations and chemicals be used to support the development of risk estimates, particularly at low exposure levels. The premise is that chromosomal alterations are involved in the development of tumors and birth defects, and that data generated for genetic alterations can be interpreted in terms of these adverse health outcomes. The general conclusions are that chromosomal alterations can be induced by ionizing radiations by a single energy loss event in a target of the size of a DNA molecule and that aberrations generally result from misrepair or failure to repair the induced lesions (generally assumed to be double-strand breaks). Chromosomal alterations induced by chemicals are produced almost exclusively by replication errors on a damaged DNA template. Thus, cell cycle stage and DNA repair and replication fidelity will be influential on overall sensitivity to aberration induction. These same features are also important in considerations of genetic susceptibility-alterations in cell cycle control or DNA repair or replication fidelity can alter sensitivity. The differences in mechanism of induction of chromosomal aberrations by ionizing radiation and chemicals is most important when considering cells at risk and comparative sensitivities among species and cell types. Models of cancer induction have gradually evolved from initiation, promotion, and progression models to multistep genetic models to the most recent one of six acquired characteristics. This evolution has passed the level of concentration of research from single gene, single cell to multiple genes (pathways), and whole tissues. The latter areas of concentration are ideal for addressing with the new genomics, proteomics, and computational modeling approaches. The attention is still on the role of genetic alterations in cancer and hereditary effects and the mechanism of their formation--it is the approaches to address these that are changing.

Animals↗

PubMed1999

Effect of hMSH6 cDNA expression on the phenotype of mismatch repair-deficient colon cancer cell line HCT15.

Mismatch recognition in human cells is mediated primarily by a heterodimer of hMSH2 and hMSH6. Cells mutated in both alleles of the hMSH6 gene are deficient in the correction of base/base mispairs and insertion/deletion loops of one nucleotide and thus exhibit a strong mutator phenotype, evidenced by elevated mutation rates and microsatellite instability, as well as by tolerance to methylating agents. The decrease in replication fidelity associated with a loss of mismatch correction implies that with each division, these cells are likely to acquire new mutations throughout their genomes. Should such secondary mutations occur in genes linked to replication fidelity or involved in the maintenance of genomic stability, they might contribute to the observed mutator phenotype. The human colon tumour line HCT15 represents one such case. Although it carries inactivating mutations in both hMSH6 alleles, it has also been shown to contain a missense mutation in the coding sequence of the proofreading domain of the polymerase-delta gene. In an attempt to find out whether the phenotype of HCT15 cells was indeed brought about solely by the lack of hMSH6, we stably transfected them with a vector carrying the wild-type hMSH6 cDNA. Our results show that although the levels of transgenic hMSH6 were low, expression of the wild-type protein resulted in a substantial restoration of mismatch binding, mismatch repair capacity and the stability of mononucleotide repeats, as well as in the reduction of mutation rates. Although methylation tolerance of the hMSH6-expressing cells was not markedly affected, the G2 cell cycle checkpoint, absent in N-methyl-N'-nitro-N-nitrosoguanidine-treated control cells, was restored.

Base Pair Mismatch↗

PubMed1993

Effects of nickel ions on polymerase activity and fidelity during DNA replication in vitro.

Nickel is a genotoxic carcinogen. However, the mechanisms of nickel-induced genotoxicity are not well understood. We have investigated the effects of Ni2+ ions on DNA polymerase activity and the fidelity of DNA replication in vitro. The effect of Ni2+ on different DNA polymerases is quite variable. The amount of enzyme inhibition and degree of alteration in replication fidelity induced by Ni2+ are dependent both on the polymerase and its associated 3'-5' exonuclease activity. Some polymerases, such as E. coli DNA polymerase I, AMV reverse transcriptase and human DNA polymerase alpha, can utilize Ni2+ as a weak substitute for Mg2+ during DNA replication. Other polymerases are very sensitive to inhibition by Ni2+ and the IC50 can vary by an order of magnitude. T4 polymerase is relatively insensitive to inhibition by Ni2+, although the sensitivity is enhanced in the absence of added Mg2+, and Ni preferentially inhibits the 3'-5' exonuclease function of T7 DNA polymerase. The fidelity and processivity of DNA polymerases may be either increased or decreased by Ni ions in a polymerase dependent manner. The inhibition DNA polymerase activity and altered replication fidelity may contribute significantly to Ni-induced mutagenesis and genotoxicity in vivo.

DNA Primers↗

PubMed2001

O-helix mutant T664P of Thermus aquaticus DNA polymerase I: altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides.

Previous studies indicate that the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant of Taq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation.

Base Sequence↗

PubMed2000

A comparison of the roles of p53 mutation and AraC inhibition in the enhancement of bleomycin-induced chromatid aberrations in mouse and human cells.

Previous studies have shown that p53 is involved in the repair of bleomycin-induced DNA damage, and that the frequency of bleomycin-induced chromatid aberrations is elevated in G(2)-treated p53 null transgenic mouse embryo fibroblasts (MEF) as compared to isogenic controls. To further characterize p53-mediated DNA repair, we studied the effect of p53 status on the ability of the DNA repair inhibitor 1-ss-D-arabinofuranosylcytosine (AraC) to sensitize MEF to bleomycin-induced chromatid aberrations. Both p53+/+ and p53-/- MEF were treated in G(2) with 0 to 7.5 microg/ml bleomycin in the presence or absence of AraC (5x10(-5) M). The frequency of bleomycin-induced chromatid aberrations was significantly higher in p53-/- cells than wild-type cells in the absence of AraC. AraC treatment significantly increased the frequency of bleomycin-induced chromatid aberrations in p53+/+ MEF to the levels in p53-/- (no AraC) but had no effect in p53-/- MEF. These results suggest that an AraC-sensitive DNA repair component is altered or absent in p53-/- cells. Similar results were observed in p53-mutant WTK1 and wild-type TK6 human lymphoblast cells exposed to 0 to 3 microg/ml bleomycin in G(2). However, AraC did cause a small increase in bleomycin sensitivity in WTK1 cells. This difference from the p53-/- MEF response may be due to differences in p53-mutant phenotype. To determine whether mutation of p53 alters DNA replication fidelity, p53+/+ and p53-/- MEF were exposed to 0 to 1 microg/ml mitomycin C (MMC). MMC did not induce chromosome aberrations in either cell line treated in G(2) but did with the same effectiveness in both cell lines treated in S-phase. Thus, p53 deficiency does not affect DNA replication fidelity or the repair of MMC-induced DNA damage.

Animals↗

PubMed1999

Effects of exonuclease activity and nucleotide selectivity of the herpes simplex virus DNA polymerase on the fidelity of DNA replication in vivo.

A mutagenesis system was developed for the in vivo study of the fidelity of DNA replication mediated by wild-type herpes simplex virus type 1 (HSV-1) strain KOS and its polymerase (Pol) mutant derivatives PAAr5, Y7, and YD12. The pHOS1 shuttle plasmid, which contained the SupF mutagenesis marker gene and the HSV oris sequence, was used for analysis of the mutation frequency and the mutation spectrum. All three Pol mutants induced significant increases in the mutation frequencies of the target gene, despite the fact that PAAr5 was previously shown to have an antimutator phenotype by the thymidine kinase mutagenesis assay (J. D. Hall, D. M. Coen, B. L. Fisher, M. Weisslitz, S. Randall, R. E. Almy, P. Gelep, and P. A. Schaffer, Virology 132:26-37, 1984; C. B. C. Hwang and J.-H. Chen, Gene 152:191-193, 1995). Altered spectra of mutated target genes induced by these three mutants were also observed. The relative frequencies of both deletion and complex mutations found in mutants induced by exonuclease-proficient Pols were significantly higher than those induced by exonuclease-deficient Pols. On the other hand, the exonuclease-deficient Pols induced significant increases in the frequency of base substitutions, which comprised predominantly G. C-to-T. A transversions, as well as mutations at additional hot spots. These results suggest that the HSV-1 DNA Pol can incorporate purine-purine or pyrimidine-pyrimidine mispaired bases which may be preferentially proofread by its intrinsic exonuclease activity. Furthermore, the effects of the sequence context of the target gene and the assay method should also be considered carefully in any analysis of replication fidelity.

Animals↗

PubMed1999

Beneficial mutations, hitchhiking and the evolution of mutation rates in sexual populations.

Natural selection acts in three ways on heritable variation for mutation rates. A modifier allele that increases the mutation rate is (i) disfavored due to association with deleterious mutations, but is also favored due to (ii) association with beneficial mutations and (iii) the reduced costs of lower fidelity replication. When a unique beneficial mutation arises and sweeps to fixation, genetic hitchhiking may cause a substantial change in the frequency of a modifier of mutation rate. In previous studies of the evolution of mutation rates in sexual populations, this effect has been underestimated. This article models the long-term effect of a series of such hitchhiking events and determines the resulting strength of indirect selection on the modifier. This is compared to the indirect selection due to deleterious mutations, when both types of mutations are randomly scattered over a given genetic map. Relative to an asexual population, increased levels of recombination reduce the effects of beneficial mutations more rapidly than those of deleterious mutations. However, the role of beneficial mutations in determining the evolutionarily stable mutation rate may still be significant if the function describing the cost of high-fidelity replication has a shallow gradient.

Alleles↗

PubMed1976

The structures and fidelity of replication of mouse mitochondrial DNA-pSC 101 EcoRI recombinant plasmids grown in E. coli K12.

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (La9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs. Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments. A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165+/-10 nucleotide pairs.

Cell Line↗

PubMed1983

Fidelity of replication of bacteriophage phi X174 DNA in vitro and in vivo.

Seven different revertants of bacteriophage phi X174am16 (AB5276G leads to T) have been isolated and the nature of the reversions determined by sequencing their DNA. The revertants each differ from am16 by just a single base substitution. These may be distinguished with varying degrees of ease by characteristic temperature sensitivities of growth. This has facilitated the determination of the frequency at which DNA polymerase III catalyses different types of substitution mutations in copying phi X174 DNA in vitro and in vivo. During the replicative form (RF) leads to single-stranded (SS) stage of replication in vitro, four different revertants may be readily produced according to well-defined rate laws on biasing the concentrations of dNTPs. Transversion mutations are found to be formed predominantly by purine x purine mismatching, whilst transitions are formed predominantly by G x T mismatching. The substitutions via G x T and G x A mismatches are estimated to occur at similar frequencies in vivo. The two most common revertants isolated in vivo, however, are not those readily produced during the RF leads to SS stage in vitro but are those produced on purine x purine mismatching in the SS leads to RF stage. The accuracy of the DNA polymerase in vitro appears to be similar to that in this stage in vivo. However, the overall accuracy of the RF leads to SS replication in vivo is more accurate than predicted from the measurements of the accuracy in vitro.

Amino Acids↗

PubMed1994

Defective mismatch repair in extracts of colorectal and endometrial cancer cell lines exhibiting microsatellite instability.

A replication error (RER+) phenotype, characterized by somatic instability in simple repeated sequences, is associated with several types of cancer. To determine if a defect in DNA replication fidelity or repair of replication errors might explain this instability, we compared both processes in cell-free extracts from RER+ endometrial and colorectal cancer cell lines to RER- cell lines. SV40 origin-dependent replication of a microsatellite sequence is highly accurate in cell extracts regardless of their RER phenotype. However, extracts from RER+ cell lines are defective in mismatch repair, while extracts of RER- cell lines are not. Lack of repair was observed when the signal (a nick) for strand-specific repair was either 3' or 5' to the mispair. One colorectal cancer cell line contained deletions in both alleles of the putative mismatch repair gene hMSH2, and one endometrial cancer cell line contained a 4-base pair duplication in one hMSH2 allele. No hMSH2 mutation was detected in the other allele or in the other five RER+ cell lines. Repair was readily detected when each of the defective extracts was mixed with a repair-proficient extract, demonstrating that no trans-acting inhibitor is present. Attempts to complement the repair deficiencies by mixing two different defective extracts identified three combinations that restored repair. The data suggest that: (i) defective repair is associated with colorectal and endometrial cancer and, by extrapolation, with other types of cancer; (ii) mutations in the hMSH2 gene, and possibly other genes, result in defective mismatch repair; (iii) the defect(s) in these lines likely involves pre-incision events or the excision step, but not the incision, polymerization, or ligation steps; and (iv) at least four functional complementation groups for mismatch repair may be involved in human cancer.

Cell Line↗