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PubMed2004

SNP discrimination through proofreading and OFF-switch of exo+ polymerase.

Single nucleotide polymorphisms (SNPs) are useful physical markers for genetic studies as well as the cause of some genetic diseases. To develop more reliable SNP assays, we examined the underlying molecular mechanisms by which deoxyribonucleic acid (DNA) polymerases with 3' exonuclease activity maintain the high fidelity of DNA replication. In addition to mismatch removal by proofreading, we have discovered a premature termination of polymerization mediated by a novel OFF-switch mechanism. Two SNP assays were developed, one based on proofreading using 3' end-labeled primer extension and the other based on the newly identified OFF-switch, respectively. These two new assays are well suited for conventional techniques, such as electrophoresis and microplates detection systems as well as the sophisticated microchips. Application of these reliable SNP assays will greatly facilitate genetic and biomedical studies in the postgenome era.

DNA Primers↗
PubMed2006

Cerulean, Venus, and VenusY67C FRET reference standards.

Förster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.

Cell Line↗
PubMed2007

Geminin in embryonic development: coordinating transcription and the cell cycle during differentiation.

Geminin was initially characterized as a bifunctional protein with roles in regulating the fidelity of DNA replication and in controlling cell fate during embryonic nervous system formation. More recently, Geminin's roles have expanded, encompassing regulation of cell proliferation and differentiation during retinogenesis, control of Hox transcription factor function during vertebrate axial patterning, and regulation of the timing of neuronal differentiation. Geminin interacts with homeodomain-containing transcription factors and with protein complexes that regulate chromatin structure, including Polycomb complexes and the catalytic subunits of the SWI/SNF chromatin remodeling complex, Brg1 and Brahma. Activities for Geminin in coordinating cellular events at the transition from proliferation to differentiation have recently emerged in multiple developmental contexts. This review will summarize Geminin's increasingly diverse roles as a developmental regulatory molecule.

Amino Acid Sequence↗
PubMed2003

Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis.

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSalpha (hMSH2 and hMSH6) and MutSbeta (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

Adenosine Triphosphatases↗
PubMed2003

On-off regulation of 3' exonuclease excision to DNA polymerization by Exo+ polymerase.

The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.

Base Sequence↗
PubMed2004

New performance from an old member: SNP assay and de novo sequencing mediated by exo+ DNA polymerases.

DNA polymerases without the 3' exonuclease function (exo(-) pol) have been widely used in sequencing and SNP genotyping. As a major player that expedited the coming of the postgenomic era, exo(-) polymerases worked remarkably well in the Human Genome Sequencing Project. However, it has become a challenge for this class of polymerases to efficiently screen the large number of SNPs that are found in the human genome. For more than three decades it has been recognized that polymerase fidelity varied according to the presence of proofreading activity that is mediated by its internal 3' exonuclease. Polymerases with proofreading function are famous for their high fidelity in DNA replication both in vivo and in vitro, but this well-known class of polymerases has been almost completely neglected in genetic analysis in the postgenomic era. We speculate that exo(+) polymerases may exhibit higher nucleotide identification ability when compared to exo- polymerases for an in vitro genetic analysis. With the application of exo(+) polymerases in SNP assays, a novel mechanism for the maintenance of DNA replication, the on/off switch, was discovered. Two new SNP assays have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of exo(+) polymerases. Furthermore, the on/off switch mechanism embodies a powerful nucleotide identification ability, which can be used to discriminate the bases that are upstream of the 3' terminus, and thus defines a new concept in de novo sequencing technology. Application of exo(+) polymerases to genetic analysis, and especially SNP assays, will greatly accelerate the pace to personalized medicine.

DNA Replication↗
PubMed1999

Microsatellite instability and p53 mutations are associated with abnormal expression of the MSH2 gene in adult acute leukemia.

Microsatellite instability (MSI) and p53 mutations have been reported to occur in a significant proportion of patients with therapy-related acute myeloid leukemia (AML). MSH2 is one of the genes involved in DNA mismatch repair to maintain fidelity of genomic replication, and defects of MSH2 are directly involved in MSI in hereditary nonpolyposis colorectal tumors and other human tumors. We have examined the expression of MSH2 protein by Western blotting in 43 adult leukemia samples, including 42 AML and 1 acute lymphoblastic leukemia (ALL) using the antibody MSH2 (Ab-1) (Calbiochem, La Jolla, CA). Abnormal expression of MSH2 protein was found in 14 of 43 (32.6%) cases; a control antibody to actin was always positive. Of the 14 patients that had abnormal expression of MSH2, 2 had therapy-related acute leukemia and 9 were elderly patients (>60 years of age). Expression of MSH2 mRNA was further examined by reverse transcriptase-polymerase chain reaction (RT-PCR). Deletion of MSH2 mRNA was found in 1 of 14 cases with deficient MSH2 protein expression. This group of patients was also screened for loss of heterozygosity (LOH) at the MSH2 locus using a panel 4 microsatellite markers (D2S367, D2S288, D2S391, and D2S2294). LOH was found in 5 of 11 cases examined. There was no evidence of LOH in 14 patients with normal MSH2 expression who were examined using the same markers. Functional evidence for defective DNA mismatch repair in leukemic cells lacking MSH2 as manifest by MSI was found in 7 of 11 cases studied. Mutations of the p53 gene in these 43 samples were also investigated by direct sequencing of full-length p53 cDNA. Mutations of p53 were found in 6 of 43 cases, including 5 of the 14 (35.7%) cases that did not express MSH2 protein. In contrast, mutation of p53 was only found in 1 of 29 (3.4%) cases with normal MSH2 protein expression (chi2 = 5.720, P <.02). These results suggest that abnormalities of DNA mismatch repair due to defective MSH2 expression could play a key role in leukemogenesis, in particular in AML arising in elderly patients or secondary to previous chemotherapy.

Acute Disease↗
PubMed2001

Highly elevated ultraviolet-induced mutation frequency in isolated Chinese hamster cell lines defective in nucleotide excision repair and mismatch repair proteins.

We have isolated N-methyl-N'-nitro-N-nitrosoguanidine-resistant cell lines from 43-3B Chinese hamster ovary cells, which are deficient in the ERCC1 gene involved in nucleotide excision repair. By Western blotting analysis, we found cell lines that are deficient or decreased in the amount of MSH6, or PMS2, or MSH2 proteins. Cell extracts of these cell lines show reduced efficiency of G:T mismatch repair activity. Compared with 43-3B, these cell lines exhibit highly elevated UV-induced mutation rates, indicating that mammalian mismatch repair can suppress UV-induced mutagenesis and may play a role in the fidelity of DNA replication at the sites of UV damage.

Adenosine Triphosphatases↗
PubMed2001

[Human Mismatch Repair System in Hematologic Malignancies]

The human mismatch repair (hMMR) system consists of members of three super-families, namely MutH, MutL and MutS. They are able to correct the error of nucleotide incorporation, increase the fidelity of DNA replication, reduce spontaneous mutation, and maintain the stability of microsatellite loci and genome. The dysfunction of MMR may induce to carcinogenesis. The relation of MMR and hematologic malignancies is presented in the article.

Journal Article↗
PubMed2004

[SOS-inducible DNA polymerases and adaptive mutagenesis].

Stability of genomes of living organisms is maintained by various mechanisms that ensure high fidelity of DNA replication. However, cells can reversibly enhance the level of replication errors in response to external factors. As mutable states are potentially involved in carcinogenesis, aging, and resistance for pathogenic agents, the existence of these states is of great importance for human health. A well-known system of inducible mutation is SOS response, whose key component is replication of damaged DNA regions. Inducible mutation implies a contribution of SOS response to the adaptation of a bacterial population to adverse environments. There is ample evidence indicating the primary role of SOS response genes in the phenomenon of adaptive mutation. The involvement of the SOS system in adaptive mutagenesis is discussed.

Adaptation, Biological↗
PubMedDate not supplied

[Evolution as resistance to entropy. I. Mechanisms of species homeostasis].

The idea is discussed that the common output of any evolution is creation of the entities that are increasingly resistant to further evolution. The moving force of evolution is entropy, the tendency to disorder. This general aspiration for chaos is a cause of the mortality of organisms and species, however, being prerequisite for any movement, it creates (by chance) novelties, which may occur (by chance) more resistant to further decay and thus survive. The surviving of those who survive is the most general principle of evolution discovered by Darwin for particular case of biological evolution. The second law of thermodynamics states that our Universe is perishing but its ontology is such that it creates resistance to destruction. The evolution is a history of this resistance. Not only those who die do not survive but also those who evolve. The entities that change (evolve) rapidly disappear rapidly and by this reason they are not observed among both the fossils and now-living organisms. We know only about long-living species. All the existing organisms are endowed with an ability to resist other changing. The following main achievements of the species homeostasis are discussed: high fidelity of DNA replication and effective mechanisms of DNA repair; diploidy; normalizing selection; truncated selection; heterozygote superiority; ability to change phenotype adaptively without changing genotype; parental care and the K-strategy of reproduction; behavior that provides independence of the environment. The global resistance of the living systems to entropy is provided the state that all the essential in biology is determined not by physical-chemical interactions but could semantic rules. A conception of "potential zygotic information" that determines the rules of ontogenesis is proposed. A zygote does not contain this information in explicit form. It is created de novo step by step during ontogenesis and it could not be decoded beforehand. The experimental data on the adaptive mutagenesis and the relevant hypothesis are discussed. It is concluded that the special mechanisms for speeding-up of evolution as created by evolution are impossible conceptually.

Adaptation, Biological↗
PubMed1991

Specificity and enzymatic mechanism of the editing exonuclease of Escherichia coli DNA polymerase III.

Exonucleolytic editing is a major contributor to the fidelity of DNA replication by the multisubunit DNA polymerase (pol) III holoenzyme. To investigate the source of editing specificity, we have studied the isolated exonuclease subunit, epsilon, and the pol III core subassembly, which carries the epsilon, theta, and alpha (polymerase) subunits. Using oligonucleotides with specific terminal mismatches, we have found that both epsilon and pol III core preferentially excise a mispaired 3' terminus and therefore have intrinsic editing specificity. For both epsilon and pol III core, exonuclease activity is much more effective with single-strand DNA; with a double-strand DNA, the exonuclease is strongly temperature-dependent. We conclude that the epsilon subunit of pol III holoenzyme is itself a specific editing exonuclease and that the source of specificity is the greater melting capacity of a mispaired 3' terminus.

Base Composition↗
PubMed1991

A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli.

Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene. To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit. The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures. The mutator effect of dnaE173 mutation is dominant over the wild type allele. These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule. DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio. We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities. However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase. Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme. It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication.

Alleles↗
PubMed1990

Fidelity of DNA polymerase alpha partially purified from a mutator mutant and wild-type mouse FM3A cells.

Several mutator mutants were isolated from a cultured mouse cell line (FM3A). The mutants exhibited a high rate of spontaneous mutation at three genetic loci of drug resistance. To investigate a possible link between mutator phenotype and fidelity of DNA replication, DNA polymerase alpha was partially purified from the wild-type and the mutator mutant (Fmut 1) showing the highest mutation rate. Using a combination of synthetic template and primer, the ratio of incorporation of incorrect to correct nucleotides was determined. The results indicated that the DNA polymerase alpha from the mutator mutant showed a slightly higher rate of misincorporation, 1.4 and 1.6 times, than that of the wild-type.

Animals↗
PubMed1994

Purified human MSH2 protein binds to DNA containing mismatched nucleotides.

The human hMSH2 protein is a member of a highly conserved family of postreplication mismatch repair components found from bacteria to humans. Alterations of the gene coding for this protein cosegregate with, and are the likely cause of, chromosome 2-linked hereditary nonpolyposis colon cancer. Postreplication mismatch repair has been found to faithfully replace misincorporated nucleotides, thereby increasing the overall fidelity of DNA replication. Loss of postreplication mismatch repair function leads to a mutator phenotype, which is proposed to account for the multiple mutations required for multistep carcinogenesis. Although the functions of hMSH2 can be anticipated based on its similarity to well-characterized bacterial and yeast proteins, proof of its functions has not been established. Here we demonstrate that purified hMSH2 binds specifically to mismatched nucleotides, providing a target for the excision repair processes characteristic of postreplication mismatch repair.

Base Sequence↗
PubMed1994

Production of a vector to facilitate DNA mutagenesis and recombination.

Most methods for the generation of site-specific mutants and recombinant constructs require primer extension in vitro. These methods include the Kunkel method and PCR-based methods. Such methods to manipulate DNA are prone to sequence error because they take place outside the complex in vivo mechanisms that increase sequence fidelity during plasmid replication in Escherichia coli. Sequence errors are of particular concern when using PCR-based methods. We have constructed two new plasmids that facilitate the generation of site-specific mutants and recombinant constructs. The plasmids we have constructed are designed to maximize the number of unique restriction enzyme recognition sites in inserts that have been cloned into either plasmid. This was accomplished by eliminating extraneous sequence and many restriction enzyme recognition sites. New recombinant circle and recombination PCR protocols for multiplex site-specific plasmid mutagenesis were used to make these plasmids. These plasmids permit small portions of an insert sequence to be readily removed by restriction enzyme digestion. A small DNA segment, containing the targeted sequence alteration, can subsequently be ligated into a plasmid construct that has not been subjected to primer extension in vitro, diminishing the probability of encountering a sequence error and reducing the amount of DNA sequencing necessary to assess for errors.

Base Sequence↗
PubMed1998

Nuclear dreams: the malignant alteration of nuclear architecture.

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets.

Animals↗
PubMed2000

Proofreading of DNA polymerase eta-dependent replication errors.

Human DNA polymerase eta, the product of the skin cancer susceptibility gene XPV, bypasses UV photoproducts in template DNA that block synthesis by other DNA polymerases. Pol eta lacks an intrinsic proofreading exonuclease and copies DNA with low fidelity, such that pol eta errors could contribute to mutagenesis unless they are corrected. Here we provide evidence that pol eta can compete with other human polymerases during replication of duplex DNA, and in so doing it lowers replication fidelity. However, we show that pol eta has low processivity and extends mismatched primer termini less efficiently than matched termini. These properties could provide an opportunity for extrinsic exonuclease(s) to proofread pol eta-induced replication errors. When we tested this hypothesis during replication in human cell extracts, pol eta-induced replication infidelity was found to be modulated by changing the dNTP concentration and to be enhanced by adding dGMP to a replication reaction. Both effects are classical hallmarks of exonucleolytic proofreading. Thus, pol eta is ideally suited for its role in reducing UV-induced mutagenesis and skin cancer risk, in that its relaxed base selectivity may facilitate efficient bypass of UV photoproducts, while subsequent proofreading by extrinsic exonuclease(s) may reduce its mutagenic potential.

Base Pair Mismatch↗