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At least 883 records · Page 49Linked to original sources
PubMed1999

RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli.

The accurate recovery of replication following DNA damage and repair is critical for the maintenance of genomic integrity. In Escherichia coli, the recovery of replication following UV-induced DNA damage is dependent upon several proteins in the recF pathway, including RecF, RecO, and RecR. Two other recF pathway proteins, the RecQ helicase and the RecJ exonuclease, have been shown to affect the sites and frequencies at which illegitimate rearrangements occur following UV-induced DNA damage, suggesting that they also may function during the recovery of replication. We show here that RecQ and RecJ process the nascent DNA at blocked replication forks prior to the resumption of DNA synthesis. The processing involves selective degradation of the nascent lagging DNA strand and it requires both RecQ and RecJ. We suggest that this processing may serve to lengthen the substrate that can be recognized and stabilized by the RecA protein at the replication fork, thereby helping to ensure the accurate recovery of replication after the obstructing lesion has been repaired.

Adenosine Triphosphatases↗
PubMed2003

Telomere instability in a human tumor cell line expressing a dominant-negative WRN protein.

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.

Carcinoma↗
PubMed1997

Mutation and haplotype analyses of the Werner's syndrome gene based on its genomic structure: genetic epidemiology in the Japanese population.

The correlation between mutations in the Werner's syndrome (WRN) gene and the haplotypes of surrounding markers was studied in Japanese patients. We have elucidated the genomic structure of WRN helicase, and found five additional mutations, designated mutations 6-10. Mutations 4 and 6 were found to be the two major mutations in this population; these mutations comprised 50.8% and 17.5%, respectively, of the total in a sample of 126 apparently unrelated chromosomes. Almost all the patients homozygous for mutation 4 shared a haplotype around the WRN gene, consistent with the view that they are derived from a single ancestor. This important advantage demonstrated in the identification of the WRN gene suggests that the Japanese present a unique population for the cloning of other disease genes. The conserved haplotype was observed across 19 loci, extending a distance estimated to be more than 1.4 Mbp around the WRN gene. This haplotype is rare among random Japanese individuals. Unexpectedly, all the nine patients homozygous for mutation 6 shared a haplotype that was identical to this haplotype at 18 of these 19 markers. These results suggest that mutations 4 and 6 arose independently in almost identical rare haplotypes. The remaining mutations (1, 5, 7, 8, 9, and 10) occurred rarely, and were each associated with different haplotypes.

DNA Helicases↗
PubMed1997

An apoptosis-inducing genotoxin differentiates heterozygotic carriers for Werner helicase mutations from wild-type and homozygous mutants.

Immortalized B lymphocytes from Werner syndrome subjects are shown to be hypersensitive to 4-nitroquinoline-1-oxide (4NQO), supporting earlier work on T lymphocytes. We also show that B cell lines from clinically normal heterozygous carriers exhibit sensitivities to this genotoxic agent, which are intermediate to those of wild-type and homozygous mutants. 4NQO is shown to induce an apoptotic response. These data encourage research on DNA repair with such cell lines and raise the question of an enhanced sensitivity of the relatively prevalent heterozygous carriers to certain environmental genotoxic agents.

4-Nitroquinoline-1-oxide↗
PubMed1999

Immunological diagnosis of Werner syndrome by down-regulated and truncated gene products.

Although immunological methods are widely used to diagnose various infectious diseases, they have rarely been employed to detect genetic diseases. In this study, we have established an immunoblot analysis system for the diagnosis of Werner syndrome (WS), a recessive genetic disorder causing premature aging and an enhanced risk of rare cancers. The method uses an immunoblot technique with specific monoclonal antibodies to WS gene product, and B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus; these cell lines express an increased level of normal WS gene product DNA helicase. The method clearly distinguishes normal from patient LCLs containing any of the mutation types found so far in Japan, primarily because of the drastically reduced levels of mutated gene products, and secondarily because of the truncated product sizes. A comparison of this immunological diagnosis with the symptom-based clinical diagnosis has narrowed down the criteria of symptoms essential for WS diagnosis. This procedure is compatible with, and has some advantage over, the genetic method, because WS patients can be diagnosed without determining the mutated gene sequences. The method exemplified in WS may also be applied to detect some other genetic diseases.

Adult↗
PubMed1997

Sequence analysis of the bovine herpesvirus type 1 genes homologous to the DNA polymerase (UL30), the major DNA-binding protein (UL29) and ICP18.5 assembly protein (UL28) genes of herpes simplex virus.

The nucleotide sequence of a 10.5 kb region (map position 0.332 to 0.410) of bovine herpesvirus type 1 (BHV-1) was determined. This region contained three open reading frames (ORFs) homologous to herpes simplex virus DNA polymerase catalytic subunit (DNApol, UL30), major DNA-binding protein (MDBP, UL29) and ICP18.5 assembly protein (ICP18.5, UL28). The BHV-1 DNApol. MDBP and ICP18.5 ORFs were 1246, 1203 and 826 amino acids long with a calculated molecular mass of 134.2 kDa, 124.4 kDa and 86.9 kDa, respectively. They showed a high homology with alphaherpesvirus homologs despite large differences in the G + C content of the UL30-UL28 segment ranging from 44.4% for varicella zoster virus to 71.5% for BHV-1. Particularly well conserved among Alphaherpesvirinae are the putative functional domains of the DNApol and MDBP proteins which are discussed. Phylogenetic analysis revealed that BHV-1 clustered in the Varicellovirus genus with the animal D-type viruses. In this group, the BHV-1 position was shown to vary according to the investigated genes. Indeed, pseudorabies virus clustered with BHV-1 in the DNApol tree but with equine herpesvirus 1 in the ICP18.5 tree.

Amino Acid Sequence↗
PubMed2001

A polymorphic variant at the Werner helicase (WRN) gene is associated with bone density, but not spondylosis, in postmenopausal women.

Werner syndrome (WS) is a rare autosomal recessive progeroid syndrome characterized by the premature onset of multiple age-related disorders. The gene responsible for WS has been identified as WRN, a member of the RecQ family of helicase genes. Based on the fact that patients with WS exhibit osteoporosis and osteoarthritis, the present study was undertaken to clarify the contribution of the WRN gene to the etiology of these two common age-related disorders in normal postmenopausal women. We investigated the association of a WRN gene polymorphism, namely c.4330 T --> C leading to an amino acid substitution from Cys to Arg, with bone density and lumbar spondylosis score in unrelated Japanese postmenopausal women (n = 377). Genotypic frequencies of T/T, T/C, and C/C were 87.5%, 12.2%, and 0.3%, respectively. Bone density of the lumbar spine (L2-4) was significantly lower in women carrying the minor C allele than in non-carriers (P = 0.037). When bone density was expressed by the Z score after being adjusted by age and weight, carriers of the C allele showed lower values not only in the lumbar spine, but also in the total body (P = 0.015 and 0.042, respectively). The association study with spondylosis in postmenopausal women (n = 221) revealed that this polymorphism was not related to the severity of spondylosis expressed by the Kellgren-Lawrence score at any disk level of the lumbar spine (L2/3-L5/S1). These findings indicate that the WRN gene may be a candidate for the genetic regulation of osteoporosis, but not spondylosis, in normal Japanese postmenopausal women.

Aged↗
PubMed2004

Molecular biology of Werner syndrome.

Human RecQ helicases, including the Werner syndrome helicase, participate in maintaining the integrity of the genome by suppressing illegitimate recombination or by repair of local DNA structural damage. Deterioration or loss of RecQ helicase associated with aging or genetic disorder results in genomic instability in tissues and organs where certain RecQ helicases are needed to correct aberrant DNA during proliferation. Such genomic instability, if not corrected, causes increased apoptotic cell death that would result in reduction of cell numbers in some tissues, leading to the deterioration of tissues and organs, the phenotypes of aging. Besides being associated with aging, genomic instability increases the risk of the development of neoplasms, both benign and malignant. These neoplasms are produced if either a checkpoint system or apoptosis is not functioning appropriately. Malignant tumor cells would then be selected from the mixed population of neoplasms by acquiring phenotypes that permit rapid cell growth and a strong capability to maintain their genome, such as tumor cells having increased levels of RecQ helicase expression, as we have observed in various tumor cell lines. Further studies are needed to discover effective measures to control genomic instability and to manage malignant tumor cells.

Animals↗
PubMed2006

Significant elevation of IgG anti-WRN (RecQ3 RNA/DNA helicase) antibody in systemic sclerosis.

Werner syndrome, caused by the homologous mutation of RecQ3 RNA/DNA helicase (WRN), is often misdiagnosed as systemic sclerosis (SSc) because of apparent similar skin changes and its relatively high frequency in Japan. The present study was undertaken to determine whether anti-WRN antibodies assayed by specific enzyme-linked immunosorbent assay occur in 41 SSc patients (30 diffuse and 11 limited types) and, if so, to determine any clinical association, such as skin sclerosis. Serum level of IgG anti-WRN antibody in SSc was significantly higher than that from 30 age- and sex-matched normal volunteers (P < 0.001). The serum level of IgG anti-WRN antibody in diffuse type SSc was significantly higher than the limited type (P < 0.05). A significant correlation was observed between serum levels of IgG anti-topoisomerase I antibody and IgG anti-WRN antibody in the same samples from SSc (P < 0.05). Moreover, in 119 normal healthy individuals aged from 0 to 99 years, a statistically significant correlation (P < 0.001) existed between serum level of IgG anti-WRN antibody and advancing age. A significantly higher level of IgG autoantibody specific for WRN detected in diffuse than in limited type SSc and normal may contribute to the pathogenesis of skin sclerosis in SSc.

Adolescent↗
PubMed2005

Polymorphisms of the TP53 codon 72 and WRN codon 1367 in individuals from Northern Brazil with gastric adenocarcinoma.

Gastric cancer is the second most frequent type of neoplasia and also the second most common cause of death in the world. TP53 codon 72, which produces variant proteins with an arginine (Arg) or proline (Pro), has been reported to be associated with cancers of the lung, oesophagus, stomach and cervix. Werner's syndrome (WS) is a premature ageing disease caused by a mutation in the WRN gene. The WRN protein acts as a DNA helicase and as an exonuclease. WRN codon 1367 produces variant proteins with an Arg or cysteine (Cys). This polymorphism has been studied, in order to understand the clinical impact of the molecular variants in WS and in age-related disorders. In the present study, the TP53 codon 72 and the WRN codon 1367 polymorphisms were investigated in 54 gastric adenocarcinoma patients (31 diffuse-type and 25 intestinal-type) and 54 controls. DNA samples were extracted, and PCR-RFLP was utilised for genotyping TP53 codon 72 and WRN codon 1367. The allele frequencies of the TP53 polymorphism were: Arg=0.74 and Pro=0.26. The allele frequencies of the WRN polymorphism were: Cys=0.73 and Arg=0.27. The crude genotypic frequencies in gastric cancer patients were similar to those of the controls, but in the WRN codon 1367 polymorphisms the mean age tended to be higher in the Arg/Arg genotypes. There also was an association, although not statistically significant, between the presence of Helicobacter pylori and the genotypes Cys/Cys and Cys/Arg and a higher percentage of cardia cancer among the Arg/Arg genotypes, and of non-cardia cancer among genotypes Cys/Cys and Cys/Arg. These findings may be a reflection of differences in the interaction between WRN codon 1367 polymorphisms and local factors in the stomach. To our knowledge, this is the first study to examine a genetic polymorphism of the WRN gene in cancer. The precise mechanisms of action of the TP53 and WRN polymorphisms involved in the aetiopathogeny of this disease need further investigation.

Adenocarcinoma↗
PubMed2005

Mutation detection in DNA oligonucleotides based on a Guanine quenching method coupled with enzymatic digestion of single-stranded DNA.

Fluorescence quenching by guanine allows DNA hybridization to be monitored and any point mutations in oligonucleotides to be detected. However, fluorescence quenching is often affected by untargeted guanine located in a protruding end (single-strand DNA) of the probe-target DNA duplex resulting in an unsatisfactory sensitivity. In the present study, we used enzymatic digestion of the protruding end of a probe-target DNA duplex to avoid interference by untargeted guanine on fluorescence quenching for detection of a nucleobase mutation. Enzymatic digestion of the protruding end of the DNA duplex fully prevented interference by untargeted guanine, and produced a marked difference in the quenching ratios (36% for wild-type, and 0% for mutant).

Base Sequence↗
PubMed1983

Anomalous electrophoretic migration of oligodeoxynucleotides with terminal -OH groups: applications for DNA exonuclease characterization.

Oligodeoxynucleotides with a terminal -OH group on both the 5' and 3' ends migrate anomalously in 23% polyacrylamide-7 M urea gels. This migration anomaly can be exploited to characterize nuclease digestion products. Thus, using specific substrates and the methods described, several types of DNA exonuclease activity can be readily distinguished.

Animals↗
PubMed1984

Replacement synthesis labeling of DNA molecules in vitro using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair.

In vitro labeling of DNA molecules using the Escherichia coli exonuclease III/DNA polymerase I enzyme pair has been examined as an alternative to existing methods of replacement synthesis labeling. It is shown that exonuclease III is able to act in a common restriction enzyme buffer [50 mM Tris (pH 8.0), 10 mM MgCl2, 50 mM NaCl] to produce a population of base-paired primer:template molecules which decrease uniformly in single-strand length with time. After heat inactivation of the exonuclease III and in the presence of radiolabeled deoxynucleotides the polymerase I reaction faithfully resynthesizes full-length molecules, asymmetrically labeled to high specific activity.

Base Composition↗
PubMed1985

Nonrandom DNA sequencing of exonuclease III-deleted complementary DNA.

The nonrandom DNA sequence analysis procedure of Poncz et al. [Proc. Natl. Acad. Sci. USA 79, 4298-4302 (1982)] was extensively modified to permit the determination of complementary DNA (cDNA) sequences containing G-C homopolymer regions. The recombinant cDNA plasmid was cleaved at a unique restriction enzyme site close to the cDNA and treated with Exonuclease III under controlled conditions to generate a set of overlapping fragments having deletions 50-1500 bases in length at the free 3' termini. After removal of single-stranded DNA regions by Bal31 and DNA polymerase I large fragment, the unique restriction enzyme site was recreated by blunt end ligation of synthetic oligonucleotides to the deleted DNA fragments and restriction enzyme digestion. The cDNA fragment was excised from the cloning vector using a second different restriction enzyme having a unique site that flanks the cDNA fragment and subsequently force-cloned into either M13 mp10 or mp11. This method should also be particularly useful for the sequencing of other types of DNA molecules with lengths 1500 bp or smaller.

Base Sequence↗
PubMed1989

A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.

We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37 degrees C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%).

Amino Acid Sequence↗
PubMed1990

Purification of eucaryotic extrachromosomal circular DNAs using exonuclease III.

A method for the isolation of eucaryotic extrachromosomal circular (ecc) DNA is described. Exonuclease III was used to preparatively digest linear and open circular forms of DNA; the resultant exonuclease-resistant molecules were then characterized by buoyant density gradient sedimentation and were found to be essentially covalently closed circular DNA. The efficiency of the exonuclease method was compared to ultracentrifugation techniques and was found to give yields greater than those obtained by two or more equilibrium density gradients. The utility of the exonuclease III technique was determined by purifying eccDNAs from mouse liver, brain, heart, and kidney tissues. The results showed that there are tissue-related differences in eccDNA content.

Animals↗