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At least 667 records · Page 37Linked to original sources
PubMed2001

A mutation in the primer grip region of HIV-1 reverse transcriptase that confers reduced fidelity of DNA synthesis.

A compensatory mutation (M230I) in the primer grip of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) restores the replication capacity of virus having a Y115W mutation in their RT coding region. The Y115W substitution impairs DNA polymerase activity and produces an enzyme with a lower fidelity of DNA synthesis. Gel-based fidelity assays with the double mutant Y115W/M230I revealed that the M230I substitution increased the accuracy of mutant Y115W. Y115W/M230I showed wild-type misinsertion fidelity in assays performed with DNA/DNA templates. However, when present alone, M230I conferred reduced fidelity as determined in misinsertion and mispair extension fidelity assays, as well as in primer extension assays carried out with three dNTPs. The mutant M230I showed a 3.3-16-fold increase in misinsertion efficiency for G, C and T opposite T, compared with the wild-type enzyme. Its fidelity was not influenced by nucleotide substitutions in the template/primer around the incorporation site. However, its accuracy was apparently affected by the structure of the 5'-overhang of the template strand. Unlike wild-type HIV-1 RT, nucleotide selectivity of mutant M230I at dT:dG, dT:dC and dT:dT mispairs was almost exclusively dependent on the K(m) values for correct and incorrect dNTPs, a characteristic that has not been described for other low fidelity mutants of HIV-1 RT.

DNA Primers↗
PubMed2002

Mechanisms by which human cells bypass damaged bases during DNA replication after ultraviolet irradiation.

The replication of damaged DNA involves cascading mechanisms of increasing complexity but decreasing accuracy. The most accurate mechanism uses low-fidelity DNA polymerases, Pol H and Pol I, which have active sites sufficiently large to accommodate a pyrimidine dimer. Replicative bypass of DNA damage by these polymerases produces an accurately replicated, newly synthesized strand. Pol H negative cells (XP-V cell lines) either adopt a proposed secondary bypass mechanism or a recombinational mode. The mechanism of the secondary bypass is unclear, but a number of experiments suggests roles for excision repair to remove damage ahead of replication forks, hRad6/18 proteolysis to clear the blocked forks, and the Rad17-RFC and 9-1-1 complexes to establish a new replication apparatus. This alternative pathway requires functional p53. In Pol H negative cells in which p53 is also inactive, the arrested fork fragments into DNA double strand breaks. Foci containing PCNA, Mre11/Rad50/Nbs1, and gamma-H2Ax can then be detected, along with chromosomal rearrangement and high frequencies of sister chromatid exchanges. The recruitment of recombination components to the arrested forks represents the ultimate failure of replication machinery to relieve the arrested state and bypass the damage. The resulting chromosomal instability in surviving cells will contribute to malignant transformation.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1988

Effect of phi X C protein on leading strand DNA synthesis in the phi X174 replication pathway.

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-base pair (bp) phi X origin of DNA replication that is the target site recognized by the phi X A protein. Removal of nucleotides from the 3' end of this 30-bp conserved origin sequence prevents the inhibitory effects of the phi X C protein. Leading strand replication of supercoiled DNA substrates containing the wild-type phi X replication origin results in the production of single-stranded circular DNA as well as the formation of small amounts of multimeric and sigma structures. These aberrant products are formed when the termination and reinitiation steps of the replication pathway reactions are skipped as the replication fork moves through the origin sequence. Replication carried out in the presence of the phi X C protein leads to a marked decrease in these aberrant structures. While the exact mechanism of action of the phi X C protein is not clear, the results presented here suggest that the phi X C protein slows the movement of the replication fork through the 30-bp origin sequence, thereby increasing the fidelity of the termination and reinitiation reactions. In keeping with the requirement for the phi X C protein for efficient packaging of progeny phi X DNA into proheads, the phi X C protein-mediated inhibition of leading strand synthesis is reversed by the addition of proteins essential for phi X bacteriophage formation. Incubation of plasmid DNA substrates bearing mutant 30 base pair phi X origin sequences in the complete packaging system results in the in vitro packaging and production of infectious particles in a manner consistent with the replication activity of the origin under study.

Bacteriophage phi X 174↗
PubMed2002

Human immunodeficiency virus type 1 recombination: rate, fidelity, and putative hot spots.

Previously, we reported that human immunodeficiency virus type 1 (HIV-1) recombines approximately two to three times per genome per replication cycle, an extremely high rate of recombination given the relatively small genome size of HIV-1. However, a recombination hot spot involving sequence of nonretroviral origin was identified in the vector system utilized, raising the possibility that this hot spot skewed the rate of recombination, and the rate of recombination observed was an overestimation. To address this issue, an HIV-1-derived vector system was used to examine the rate of recombination between autologous HIV-1 sequences after restricting replication to a single cycle in the absence of this hot spot. Viral DNA and RNA were analyzed by a combination of the heteroduplex tracking assay, restriction enzyme analysis, DNA sequencing, and reverse transcription-PCR. The results indicate that HIV-1 undergoes recombination at a minimum rate of 2.8 crossovers per genome per cycle. Again, this is a very high rate given the small size of the HIV-1 genome. The results also suggested that there might be local hot spots of recombination at different locations throughout the genome since 13 of the 33 strand transfers identified by DNA sequencing shared the same site of recombination with one or two other clones. Furthermore, identification of crossover segments also allowed examination of mutations at the point of recombination, since it has been predicted from some studies of cell-free systems that mutations may occur with a frequency of 30 to 50% at crossover junctions. However, DNA sequence analysis of crossover junctions indicated that homologous recombination during viral replication was not particularly mutagenic, indicating that there are other factors or conditions not yet reproduced in cell-free systems which contribute to fidelity during retroviral recombination.

Cell Line↗
PubMed1999

The fidelity of template-directed oligonucleotide ligation and the inevitability of polymerase function.

The first living systems may have employed template-directed oligonucleotide ligation for replication. The utility of oligonucleotide ligation as a mechanism for the origin and evolution of life is in part dependent on its fidelity. We have devised a method for evaluating ligation fidelity in which ligation substrates are selected from random sequence libraries. The fidelities of chemical and enzymatic ligation are compared under a variety of conditions. While reaction conditions can be found that promote high fidelity copying, departure from these conditions leads to error-prone copying. In particular, ligation reactions with shorter oligonucleotide substrates are less efficient but more faithful. These results support a model for origins in which there was selective pressure for template-directed oligonucleotide ligation to be gradually supplanted by mononucleotide polymerization.

Base Sequence↗
PubMed2002

Nuclear mitochondrial interplay in the modulation of the homopolymeric tract length heteroplasmy in the control (D-loop) region of the mitochondrial DNA.

We have studied the genetic characteristics of a homopolymeric tract length heteroplasmy associated with the 16189C variant in the mtDNA D-loop control region to identify the factor(s) involved in the generation of the length heteroplasmy. The relative proportion of the various lengths of the polycytosines (i.e., the pattern of the length heteroplasmy) is maintained in an individual, and previous evidence shows that it is regenerated de novo following cell divisions. The pattern is maintained in maternally related individuals, suggestive of mtDNA determinants. Of the 38 individuals with the 16189C variant studied, 39% were found to exhibit the (16180)AAACCCCCCCCCCC(16193) variant associated with A16183C polymorphism [(11C)-group], while 53% showed the (16180)AACCCCCCCCCCCC(16193) variant associated with a further A16182C polymorphism [(12C)-group]. Haplotype analysis of the mtDNA revealed a specific association of the longer mean length of the poly[C] in the (12C)-group with haplogroup B. A similar association was also observed in the (11C)-group, but with a novel haplogroup. Cybrid constructions revealed that the involvement of nuclear factor(s) in the generation of the length heteroplasmy is prominent in homopolymeric tract of eight cytosines. The nuclearly coded factor(s) is/are presumably related to the fidelity of the nuclearly coded components of the mitochondrial DNA replication machinery.

Base Sequence↗
PubMed1987

RecA protein and SOS. Correlation of mutagenesis phenotype with binding of mutant RecA proteins to duplex DNA and LexA cleavage.

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. We have suggested that RecA might participate directly in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g. pyrimidine dimer) because of its partially unwound nature; DNA polymerase III will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site more often but with reduced fidelity. In support of this proposal, we have found that the phenotype of wild-type and mutant RecA for mutagenesis correlates with capacity to bind to double-stranded DNA. Wild-type RecA binds more efficiently to ultraviolet (u.v.)-irradiated, duplex DNA than to non-irradiated DNA. The RecA441 (Tif) protein that is constitutive for mutagenesis binds extremely well to double-stranded DNA with no lesions, whereas the RecA430 protein that is defective in mutagenesis binds poorly even to u.v.-irradiated DNA. The RecA phenotype also correlates with capacity to use duplex DNA as a cofactor for cleavage of the LexA repressor protein for SOS-controlled operons. Wild-type RecA provides efficient cleavage of LexA only with u.v.-irradiated duplex DNA; RecA441 cleaves well with non-irradiated DNA; RecA430 gives very poor cleavage even with u.v.-irradiated DNA. We conclude that the interaction of RecA with damaged double-stranded DNA is likely to be a critical component of SOS mutagenesis and to define a pathway for the LexA cleavage reaction as well.

Bacterial Proteins↗
PubMed1996

Mutational spectra vary with exposure conditions: benzo[a]pyrene in human cells.

The AHH-1 human lymphoblastoid line was exposed to benzo[a]pyrene under markedly different conditions: a single toxic exposure of 30 microM for 28 h, a nontoxic exposure of 0.5 microM for 6 days and an exposure approximating estimates of BP concentration in the human lung of 20 nM for 20 days. Duplicate cultures containing 2 x 10(9) cells each were used to assure the statistical quality of the mutational spectra. Point mutational hotspots were observed in bp 215 to 318 of the third exon of the hprt gene after mutants were selected en masse with 6-thioguanine, using a combination of denaturing gradient gel electrophoresis and high fidelity polymerase chain reaction. The spectra were highly reproducible in replicate experiments but varied dramatically among treatment conditions. These data demonstrate that mutational spectra were critically dependent upon conditions of exposure. The results significantly extend prior reports on this subject and clarify an important issue for the use of mutational spectra obtained in vitro to create hypotheses about what spectra may be expected in humans in vivo. We conclude that commonly used protocols of short-term exposure to mutagenic chemicals at high concentrations should not be used to define such expectations. Rather, the more difficult protocols of long-term and low-concentration mutation studies are justified as conditions necessary, although perhaps not sufficient, to approximate human in vivo mutational pathways.

B-Lymphocytes↗
PubMed2005

DNA polymerases and somatic hypermutation of immunoglobulin genes.

Somatic hypermutation of immunoglobulin variable genes, which increases antibody diversity, is initiated by the activation-induced cytosine deaminase (AID) protein. The current DNA-deamination model posits that AID deaminates cytosine to uracil in DNA, and that mutations are generated by DNA polymerases during replication or repair of the uracil residue. Mutations could arise as follows: by DNA replicating past the uracil; by removing the uracil with a uracil glycosylase and replicating past the resulting abasic site with a low-fidelity polymerase; or by repairing the uracil and synthesizing a DNA-repair patch downstream using a low-fidelity polymerase. In this review, we summarize the biochemical properties of specialized DNA polymerases in mammalian cells and discuss their participation in the mechanisms of hypermutation. Many recent studies have examined mice deficient in the genes that encode various DNA polymerases, and have shown that DNA polymerase H (POLH) contributes to hypermutation, whereas POLI, POLK and several other enzymes do not have major roles. The low-fidelity enzyme POLQ has been proposed as another candidate polymerase because it can efficiently bypass abasic sites and recent evidence indicates that it might participate in hypermutation.

Animals↗
PubMed2000

Homologous recombination as a mechanism for genome rearrangements: environmental and genetic effects.

Novel findings over the last 2 years have led to an increased emphasis on homologous recombination (HR) as both a pathway for DNA repair and a cause for genomic rearrangements. Indeed, environmental carcinogens increase the frequency of HR, as can be observed when two copies of a duplicated sequence recombine to delete the intervening sequences. Such HR events between dispersed homologous sequences may result in not only deletions, but also gene duplications or translocations. These types of genomic rearrangement have been observed to be the cause of several different genetic diseases, including cancer. In reflection of this, several genes have been identified that, when mutant, predispose an individual to an increased frequency of cancer. These genes have been shown to be either directly or indirectly involved in HR. In addition, HR is induced by a wide variety of carcinogens, preferentially in proliferating cells. This fits the most current models of recombination and its involvement in reinitiating stalled replication forks. Thus, 'correct' HR repair may act with high fidelity, an important issue for proliferating cells, but in the context of alternative homologous partner sequences, 'aberrant' HR can cause genomic rearrangements with dire consequences.

Animals↗
PubMed2001

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion.

CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.

Animals↗
PubMed2004

Enhancer trapping in zebrafish using the Sleeping Beauty transposon.

BACKGROUND: Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem. RESULTS: We modulated a Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline. CONCLUSIONS: This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome.

Animals↗
PubMed1981

Recent research on nickel carcinogenesis.

Research on nickel carcinogenesis from 1975 to March 1980 is reviewed. Epidemiological studies have strengthened the evidence that workers in nickel refineries have increased risks of cancers of the nasal cavities and lungs. Clinical investigations have resulted in improved diagnosis, classification, and management of cancers of respiratory organs in nickel refinery workers. Carcinogenicity tests have demonstrated the carcinogenicity of nickel subsulfide (alpha-Ni3S2) in rodents following administration by a variety of parenteral routes. Radiotracer studies and x-ray diffractometry have clarified the metabolism of alpha-Ni3S2 in rodents. In vitro exposures of mammalian cells to certain nickel compounds have been shown to inhibit cellular uptake of thymidine-3H, and to induce chromosomal aberrations, somatic mutations, and morphological transformation. Mutagenicity tests of nickel compounds in bacterial systems have consistently been negative. Ni(II) has beeb reported to impair the fidelity of viral and bacterial DNA polymerases for in vitro replication of synthetic nucleotide templates.

Administration, Oral↗
PubMedDate not supplied

A case reopened: teleology and its consequences for the units of selection discussion.

Darwinian explanations for teleology are often imprecise, and justify the occurrence of teleological features by referring to natural selection in a vague and unspecified sense. In this paper, the Darwinian account for teleology is further analyzed. It is argued that in theory only a specific form of teleology--teleology that is caused by and directed towards the preservation of the genetic program--can be explained in a naturalistic way by employing Darwin's theory of natural selection. This observation links teleology with the units of selection discussion, as for both discussions the end-direction of teleological processes and behavior is of elementary importance. According to Dawkins' analysis, the unit of selection is an active germ-like replicator with a sufficient degree of longevity-fecundity-copying fidelity. From the teleological point of view, the unit of selection should additionally incorporate the genetic program in order to naturalize teleology. It is shown that within sexually reproducing species these two requirements cannot be met. Dawkins' concept of genic selectionism cannot be maintained without violating the naturalistic claims on teleology, and none of the other frequently proposed unit of selection candidates can adequately meet the demands as developed by Dawkins and those developed in the light of teleology.

Adaptation, Physiological↗
PubMed1998

Fanconi anemia C gene product plays a role in the fidelity of blunt DNA end-joining.

Mutations in genes controlling the correct functioning of the replicative, repair and recombination machineries may lead to genomic instability. A high level of spontaneous chromosomal aberrations amplified by treatment with DNA cross-linking agents is the hallmark of Fanconi anemia (FA), an inherited chromosomal instability syndrome associated with cancer proneness. Two of the eight FA genes have been cloned (FAA and FAC), but their function has not yet been defined. The lack of homology with known genes suggests the involvement of FA genes in a novel pathway specific to vertebrates. Using a DNA end-joining assay in cultured cells, we studied the processing of both blunt and cohesive-ended double strand breaks (DSB) in normal and FA cells. The results show that: (i) the overall ligation efficiency is normal in FA lymphoblasts; (ii) in FA-C, error-free processing of blunt-ended DSB is markedly decreased, resulting in a higher deletion frequency and larger deletion size; (iii) the fidelity of processing of blunt-DSB is completely restored in FACC cells (complemented with wild-type FAC gene) and the deletion size shifted to values similar to that observed in normal cells; (iv) the fidelity of cohesive end-joining is not affected in FA cells; (v) activities and/or expression of known factors involved in DSB processing, such as the components of the DNA-PK complex and XRCC4, are normal in FA cells. Our results provide strong evidence that the lack of a functional FAC gene results in loss of fidelity of end-joining, which likely accounts for the FA-C phenotype of chromosome instability. We conclude that FAC, and perhaps all FA gene products, are likely to play a role in the fidelity of end-joining of specific DSB.

Amino Acid Sequence↗
PubMed1981

Metal-induced infidelity of DNA synthesis.

A number of metals have been demonstrated to be mutagens in procaryotic and eucaryotic organisms as well as carcinogens in experimental animals. Epidemiologic studies have indicated that Ni, Cr, and As are involved in human carcinogenesis. We have hypothesized that the active molecular species is the cation and that metal induced mutations result from incorrect base-substitutions during DNA replication. This is supported by the observations that metal ions diminish the fidelity of DNA synthesis in vitro using a variety of DNA polymerases. There is a significant correlation between the metals that decrease fidelity and those that have been reported to be mutagenic and carcinogenic. Thus, metal carcinogens are no exception to the general postulate that carcinogens can be identified by their effects on DNA.

Animals↗
PubMed2005

Many players, one goal: how chromatin states are inherited during cell division.

Replication of genomic material is a process that requires not only high fidelity in the duplication of DNA sequences but also inheritance of the chromatin states. In the last few years enormous effort has been put into elucidating the mechanisms involved in the correct propagation of chromatin states. From all these studies it emerges that an epigenetic network is at the base of this process. A coordinated interplay between histone modifications and histone variants, DNA methylation, RNA components, ATP-dependent chromatin remodeling, and histone-specific assembly factors regulates establishment of the replication timing program, initiation of replication, and propagation of chromatin domains. The aim of this review is to examine, in light of recent findings, how so many players can be coordinated with each other to achieve the same goal, a correct inheritance of the chromatin state.

Adenosine Triphosphate↗
PubMed1998

Program fidelity in assertive community treatment: development and use of a measure.

Assertive community treatment (ACT) is a complex community-based service approach to helping people with severe mental disorders live successfully in the community. Effective replication of the model and research on critical elements require explicit criteria and measurement. A measure of program fidelity to ACT and the results of its application to fifty diverse programs are presented.

Community Mental Health Services↗