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At least 199 records · Page 11Linked to original sources
PubMed1996

Analysis for microsatellite instability and mutations of the DNA mismatch repair gene hMLH1 in familial gastric cancer.

We examined 30 gastric-cancer patients with a varying degree of family history of stomach cancer and/or synchronous gastric tumors for microsatellite instability. We observed microsatellite instability at at least 1 of 8 loci tested in tumors of 14/30 patients; of these 14, 8 had single locus alterations and 6 had alterations at at least half of the 8 loci. Among the patients with microsatellite instability at > or = 4 loci, 3 patients showed a strong familial clustering of gastric cancer. Mutation analysis of the DNA mismatch repair gene hMLHl on paired non-tumorous and tumor DNA from 10 patients, 6 with microsatellite instability at > or = 4 loci and 4 with an alteration at one locus, revealed a novel missense mutation, present in the normal and tumor DNA of one patient with microsatellite instability at multiple loci in his tumor. His family history of cancer included one second-degree relative affected with gastric cancer. These data suggest that germline mutations in the hMLHl gene occur in some gastric-cancer patients and that in the majority of cases microsatellite instability in gastric tumors may be due to defects in other genes responsible for DNA replication fidelity than the hMLHl.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

Global analysis of lymphocyte gene expression: perturbation of H-9 cells by infection with distinct isolates of human immunodeficiency virus--an exposition by multivariate analysis of a host-parasite interface.

AIDS is a progressive disease associated with steady loss of helper T cells and several other functions. As the disease evolves, cytopathogenic human immunodeficiency (HIV) variants of increasing virulence can be isolated from the host. The HIV is an unusually variable genome by virtue of a low replication fidelity. In this report we describe our effort to test the hypothesis that there is a correlation between virus variability and cytopathogenicity, and further, that there is an "impact" of the virus infection on the expression of host cellular genes. To search for such a relationship, we infected H-9 cells (human CD4+ lymphoblastoid cell line) with each of 5 isolates of HIV of distinct origin and cytopathogenicity. To measure the influence of the virus infection on the expression of host cellular genes, shortly after infection, (3 h or 13 h), cells were radiolabeled and the radioactive polypeptides separated by two-dimensional gel electrophoresis. Radiofluorographs were prepared and analyzed to determine relative rates of biosynthesis of cellular polypeptides. To organize the large amounts of data found, cluster analysis and principal component analysis were used to expose the data in formats that allowed a model construction. The rates of biosynthesis of many cellular polypeptides were altered upon viral infection in terms of both enhancements and impairment of biosynthesis. Some of the variation in polypeptide synthesis was isolate-specific, while most alterations were of modest magnitude. There appears to be no "overall effect" associated with infection by a cytopathic variant of the virus. Polypeptides affected by the cytopathic variants were determined as targets for further investigation. The method used promotes the measurement of "ensemble" information that is characteristic of the process and it promotes the creation of models of virus action.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Characterization of the nuclear import of human MutLalpha.

DNA mismatch repair (MMR) is essential for the maintenance of replication fidelity. Its major task is to recognize mismatches as well as insertion/deletion loops of newly synthesized DNA strands. Although different players of human MMR have been identified, the regulation of essential steps of MMR is poorly understood. Because MMR is initiated in the nucleus, nuclear import might be a mechanism to regulate MMR. Nuclear targeting is accomplished by conserved signal sequences called nuclear localization signals (NLS), which represent clusters of positively charged amino acids (aa). hMLH1 contains two clusters of positively charged amino acids, which are candidate NLS sequences (aa 469-472 and 496-499), while hPMS2 contains one (aa 574-580). To study the effect of these clusters on nuclear import, NLS mutants of hMLH1 and hPMS2 were generated and expressed in 293T cells. The subcellular localization of the mutant constructs was monitored by confocal laser microscopy. We demonstrated that missense mutations of two signal sequences, one in hMLH1 and one in hPMS2, lead to impaired nuclear import, which was especially prominent for mutants of the hMLH1 residues K471 and R472; and hPMS2 residues K577 and R578.

Amino Acid Sequence↗
PubMed2000

Phage phi 29 DNA polymerase residues involved in the proper stabilisation of the primer-terminus at the 3'-5' exonuclease active site.

Three highly conserved amino acid residues have been characterised here as ssDNA ligands at the 3'-5' exonuclease active site of o29 DNA polymerase. The functional role of Tyr59, His61 and Phe69 residues of o29 DNA polymerase (belonging to Exo II motif, previously described as containing an invariant catalytic aspartate residue and two highly conserved ssDNA ligands) was assayed by biochemical analysis of six site-directed mutants at those residues. These studies revealed that the mutations introduced severely affected their ssDNA binding capacity and, as a consequence, the 3'-5' exonuclease activity on ssDNA substrates was also severely impaired, producing drastic defects in the maintenance of replication fidelity. Crystal structures of Klenow fragment of Pol Ik and Thermococcus gorgonarius DNA polymerase complexed with ssDNA at their 3'-5' exonuclease active sites revealed that residues Gln419 of the former, and Tyr209 of the latter, the counterparts of His61 of o29 DNA polymerase, are making contacts with the penultimate phosphodiester bond of ssDNA substrate. Here, the functional role of this residue is described.

Amino Acid Motifs↗
PubMed1987

Mutagenic DNA repair in Escherichia coli. XIV. Influence of two DNA polymerase III mutator alleles on spontaneous and UV mutagenesis.

We introduced the dnaE486 and polC74 mutations (which are associated with decreased DNA polymerase III replication fidelity) into excision defective Escherichia coli strains with varying SOS responses. These mutations increased the UV-induced frequency of base pair substitution mutations in all strains tested, except recA430 and umuC122 derivatives. This UV mutator effect therefore requires expression of the SOS error-prone repair system. In recA441 lexA51 strains where the SOS system is constitutively expressed, the UV mutator effect of the dnaE alleles was similar in relative terms (though greater in absolute terms). Since these dnaE alleles decrease rather than increase survival after UV it is argued that they promote a burst of untargeted mutations close to UV photoproducts ("hitch-hiking" mutations) rather than increase the number of translesion synthesis events. The fact that there was no UV mutagenesis in dnaE486 umuC122 or polC74 umuC122 strains indicates that infidelity associated with these dnaE alleles did not of itself enable translesion synthesis to occur. The spontaneous mutator effect conferred by dnaE486 and polC74 was not affected by umuC122 or recA430 indicating that it is not dependent upon error-prone repair ability. In recA441 lexA51 bacteria, where SOS error-prone repair is constitutively induced, the mutator effect of dnaE486 was greater and was largely blocked by umuC122. It is suggested that spontaneously occurring cryptic lesions that are themselves unable to induce the SOS system are subject to translesion synthesis under these conditions and trigger a burst of hitch-hiking mutations that are therefore effectively umuC dependent.

Alleles↗
PubMed1987

The evolution of multicompartmental genomes in viruses.

The genetic information of many viruses is divided between separately encapsidated nucleic acid molecules. A simple evolutionary model is constructed to explain this phenomenon. All multicompartmental viruses infect plants, and most are RNA viruses. The former fact may be due to the high transmission multiplicities enjoyed by plant viruses. The latter may be due to the low replication fidelity of RNA, although another explanation is also offered. The logic of the analysis is contrasted with that of previous explanations. In particular, this paper proceeds from a "selfish DNA" viewpoint. It is not necessary to suppose that the division of the genome fills any adaptive function for the virus. The theory makes testable predictions about the parameters of multicompartmental viruses.

Biological Evolution↗
PubMed2006

DNA mismatch repair and Lynch syndrome.

The evolutionary conserved mismatch repair proteins correct a wide range of DNA replication errors. Their importance as guardians of genetic integrity is reflected by the tremendous decrease of replication fidelity (two to three orders of magnitude) conferred by their loss. Germline mutations in mismatch repair genes, predominantly MSH2 and MLH1, have been found to underlie the Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), a hereditary predisposition for cancer. Lynch syndrome affects predominantly the colon and accounts for 2-5% of all colon cancer cases. During more than 30 years of biochemical, crystallographic and clinical research, deep insight has been achieved in the function of mismatch repair and the diseases that are associated with its loss. We review the biochemistry of mismatch repair and also introduce the clinical, diagnostic and genetic aspects of Lynch syndrome.

Adenosine Triphosphatases↗
PubMed2006

Layered double hydroxide minerals as possible prebiotic information storage and transfer compounds.

One of the fundamental difficulties when considering the origin of life on Earth is the identification of an emergent system that not only replicated, but also had the capacity to undergo discrete mutation in such a way that following generations might inherit and pass on the mutation. We speculate that the layered double hydroxide (LDH) minerals are plausible candidates for a proto-RNA molecule. We describe a hypothetical LDH-like system which, when intercalated with certain anions, forms crystals with a high degree of internal order giving rise to novel information storage structures in which replication fidelity is maintained, a concept we use to propose an explanation for interstratification in terephthalate LDHs. The external surfaces of these hypothetical crystals provide active sites whose structure and chemistry is dictated by the internal information content of the LDH. Depending on the LDH polytype, the opposing external surfaces of a crystal may give rise to reactive sites that are either complementary or mirror images of each other, and so may be chiral. We also examine similarities between these proposed "proto-RNA" structures and the DNA that encodes the hereditary information in life today, concluding with a hypothetical scenario wherein these proto-RNA molecules predated the putative RNA-world.

Hydroxides↗
PubMed1984

Generation of genetic diversity in herpes simplex virus: an antimutator phenotype maps to the DNA polymerase locus.

We have measured the spontaneous production of mutants in derivatives of herpes simplex virus type 1 resistant to phosphonoacetic acid. Six such derivatives produced 9- to 123-fold fewer iododeoxycytidine (ICdR-)-resistant progeny (i.e., thymidine kinase deficient) than their wild-type parents. To locate the mutation which controls mutant production in one of the strains (PAAr-5), we constructed phosphonoacetic acid-resistant, recombinant viruses by marker transfer, using wild-type viral DNA and DNA restriction fragments conferring the resistance phenotype. The resultant recombinants also produced very low levels of ICdR-resistant progeny during growth, indicating a close linkage (within 1.1 kilobase pairs) between the drug resistance locus and the sequences controlling production of mutant progeny. Evidence is presented that the low mutant yield in PAAr-5 is not due to abnormal expression of mutants, hypersensitivity to ICdR, altered thymidine kinase activity, or slow replication rates. Since the locus conferring resistance to phosphonoacetic acid in PAAr-5 has been shown previously to be the DNA polymerase gene, we hypothesize that the reduced yield of mutants results from enhanced replication fidelity by the altered DNA polymerase. The existence of antimutator derivatives of herpes simplex indicates that the observed high mutation rate for wild-type strains is an intrinsic property of the virus and may provide a selective advantage during growth in animal hosts.

Cell Line↗
PubMed1994

Landscapes: complex optimization problems and biopolymer structures.

The evolution of RNA molecules in replication assays, viroids and RNA viruses can be viewed as an adaptation process on a 'fitness' landscape. The dynamics of evolution is hence tightly linked to the structure of the underlying landscape. Global features of landscapes can be described by statistical measures like number of optima, lengths of walks and correlation functions. The evolution of a quasispecies on such landscapes exhibits three dynamical regimes depending on the replication fidelity: Above the "localization threshold" the population is centered around a (local) optimum. Between localization and "dispersion threshold" the population is still centered around a consensus sequence, which, however, changes in time. For very large mutation rates the population spreads in sequence space like a gas. The critical mutation rates separating the three domains depend strongly on characteristics properties of the fitness landscapes. Statistical characteristics of RNA landscapes are accessible by mathematical analysis and computer calculations on the level of secondary structures: these RNA landscapes belong to the same class as well known optimization problems and simple spin glass models. The notion of a landscape is extended to combinatory maps, thereby allowing for a direct statistical investigation of the sequence structure relationships of RNA at the level of secondary structures. Frequencies of structures are highly non-uniform: we find relatively few common and many rare ones, as expressed by a generalized form of Zipf's law. Using an algorithm for inverse folding we show that sequences sharing the same structure are distributed randomly over sequence space. Together with calculations of structure correlations and a survey of neutral mutations this provides convincing evidence that RNA landscapes are as simple as they could possibly be for evolutionary adaptation: Any desired secondary structure can be found close to an arbitrary initial sequence and at the same time almost all bases can be substituted sequentially without ever changing the shape of the molecule. Consequences of these results for evolutionary optimization, the early stages of life, and molecular biotechnology are discussed.

Adaptation, Biological↗
PubMedDate not supplied

Influence of recF on spontaneous mutation in Escherichia coli.

A recF mutation increased the spontaneous mutation rate for mutations to histidine and tryptophan independence, and to streptomycin resistance but not significantly to rifampicin resistance. The results suggest the recF gene may have a role in maintaining DNA replication fidelity.

Bacterial Proteins↗
PubMed2026

Functions and mechanisms of BRCA1 in early embryonic development.

Breast Cancer Gene 1 (BRCA1) is a critical regulator of genome integrity whose dysfunction greatly increases lifetime risk of breast and ovarian cancers. While BRCA1 has been extensively studied in the contexts of adult biology and cancer, its diverse functions, including homologous recombination-mediated DNA repair, cell cycle checkpoint activation, protein ubiquitination, and transcriptional regulation, have many underexplored implications. In early embryonic development, the maternal-to-zygotic transition (MZT) and subsequent developmental processes place extraordinary demands on DNA replication fidelity, cell cycle regulation, transcriptional activation, and chromatin remodeling. These critical processes overlap strikingly with canonical functions of BRCA1, yet its function in early development is poorly characterized. In this review, we investigate BRCA1 conservation across species and connect its well-established functions to findings from developmental studies to assess its role in development. We highlight evidence of BRCA1 mitigating genome integrity loss from diverse sources, maintaining the proliferative activity needed for successful germ layer formation and early tissue morphogenesis, and regulating transcription and epigenetic modifications. Together, this synthesis supports a model where BRCA1 acts as a multi-functional and dynamic regulator of early embryogenesis. Building on this, we propose outstanding questions that could further illuminate these developmental roles. Characterization of BRCA1 in early development may not only provide important insight into the origin and progression of cancer susceptibility but may also elucidate fundamental mechanisms shaping early development.

BRCA1 Protein↗
PubMed2004

Pretumor progression: clonal evolution of human stem cell populations.

Multistep carcinogenesis through sequential cycles of mutation and clonal succession is usually described as tumor progression, or the clonal evolution of tumor cell populations. However, many mutations found in cancers are also compatible with normal appearing phenotypes and therefore genetic progression may precede tumor progression. To better characterize such pretumor progression (mutations in the absence of visible phenotypic changes), a quantitative model was developed that postulates most oncogenic cancer mutations first accumulate in normal appearing colon crypt niche stem cells. Each crypt contains multiple stem cells, and random niche stem cell loss with replacement eventually leads to the loss of all stem cell lineages except one. This niche succession or crypt clonal evolution is similar to the clonal succession of tumor progression except it does not require selection or change visible phenotype. Mutations may sequentially accumulate during stem cell clonal evolution either through drift (passenger mutations) or selection. To determine the feasibility of pretumor progression, mutation rates sufficient to recreate the epidemiology of colorectal cancer were estimated. Pretumor progression may completely substitute for visible tumor progression because it is theoretically possible for all cancer mutations to first accumulate in normal appearing colon with normal replication fidelity. Elevated mutation rates or tumorigenesis may be unnecessary for early progression.

Adult↗
PubMed2004

Colorectal pretumor progression before and after loss of DNA mismatch repair.

A pretumor progression model predicts many oncogenic cancer mutations may first accumulate in normal appearing colon. Although direct observations of early pretumor mutations are impractical, it may be possible to retrospectively reconstruct tumor histories from contemporary cancer mutations. To infer when and in what order mutations occur during occult pretumor progression, we examined 14 cancers from individuals with heterozygous germline mutations in DNA mismatch repair (MMR) genes or hereditary nonpolyposis colorectal cancer (HNPCC). Somatic inactivation of the normal allele occurs sometime during a lifetime and results in loss of MMR, elevated mutation rates, and subsequent widespread somatic microsatellite mutations in HNPCC cancers. Patient ages at MMR loss can be estimated because intervals between MMR loss and cancer removal can be inferred from numbers of microsatellite tumor mutations. The relative order of MMR loss during pretumor progression may also be inferred from its collective ages of occurrence. Somatic MMR loss preceded cancer removal by an average of 6.1 years, occurred relatively late in life (average of 41.6 versus 47.7 years at cancer removal), and was a surprisingly late (fifth or sixth) step. Calculations indicate five or six oncogenic mutations could accumulate with relatively normal replication fidelity in normal appearing colon. HNPCC pretumor progression essentially begins from birth and ends with MMR loss, implying elevated mutation rates and tumorigenesis may be unnecessary for most progression.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Damage repair DNA polymerases Y.

The newly found Y-family DNA polymerases are characterized by low fidelity replication using an undamaged template and the ability to carry out translesion DNA synthesis. The crystal structures of three Y-family polymerases, alone or complexed with DNA and nucleotide substrate, reveal a conventional right-hand-like catalytic core consisting of finger, thumb and palm domains. The finger and thumb domains are unusually small resulting in an open and spacious active site, which can accommodate mismatched base pairs as well as various DNA lesions. Although devoid of a 3'-->5' exonuclease activity, the Y-family polymerases possess a unique "little finger" domain that facilitates DNA association, catalytic efficiency and interactions with auxiliary factors. Expression of Y-family polymerases is often induced by DNA damage, and their recruitment to the replication fork is mediated by beta-clamp, clamp loader, single-strand-DNA-binding protein and RecA in Escherichia coli, and by ubiquitin-modified proliferating cell nuclear antigen in yeast.

Binding Sites↗
PubMed1999

Escherichia coli mutator genes.

The isolation and characterization of Escherichia coli mutator genes have led to a better understanding of DNA replication fidelity mechanisms and to the discovery of important DNA repair pathways and their relationship to spontaneous mutagenesis. Mutator strains in a population of cells can be beneficial in that they allow rapid selection of variants during periods of stress, such as drug exposure.

DNA Repair↗
PubMed2003

Polymorphism of genes encoding SOS polymerases in natural populations of Escherichia coli.

High fidelity replicative DNA polymerases can be blocked during DNA replication by various base damages, which represents a potentially lethal event. Escherichia coli possesses three DNA polymerases, PolII, PolIV and PolV, that can continue replication over such lesions in template DNA, thus allowing for cell survival. Genes coding for these enzymes, polB, dinB, and umuCD respectively, belong to the stress-inducible SOS regulon. We have analyzed the patterns of nucleotide sequence variability of genes encoding for three SOS polymerases from E. coli natural isolates in order to identify the nature of selective forces that determine their evolution. The frequency of inferred inter-strain recombination events, and the frequency of synonymous and non-synonymous base substitutions within these genes do not deviate significantly from those observed for the control group composed of 2 genes coding for DNA polymerases PolI and PolIII and 10 metabolic genes. This suggests that the loci coding for SOS polymerases are subject to selective pressure for the maintenance of their function and specificity. The fact that genes coding for translesion-synthesis (TLS) polymerases, particularly dinB and umuC homologs, have been conserved during evolution and the present analysis suggest that their activity is essential for the cellular survival and fitness.

Bacterial Proteins↗
PubMed1992

Minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment).

The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.

Base Sequence↗