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PubMed1981

Inhibitor studies of phage T4 wild-type and mutant DNA polymerases. V. A. summary of kinetic and inhibitor data.

The DNA polymerases of phage T4 wild-type, the mutator mutant L98 and the antimutator mutant CB121 were purified about 100-fold free of foreign enzyme activities interfering with the polymerase assay. The enzymes were characterized as to thermostability, exonuclease activity and kinetic data with DNA template primer, deoxythymidine 5' -triphosphate, and a mixture of all four deoxynucleoside 5' -triphosphates. The effects of eight inhibitors of DNA synthesis on the three enzymes were determined (Schroeder and Jantschak 1978 and 1980, Jantschak and Schroeder 1980) and are compared here. The most selective inhibitor, pyridoxal 5' -phosphate, interacts with the active site of the polymerases while the two least discriminating drugs, distamycin A and actinomycin D, do not directly interact with the polymerases at all. The study was intended to test whether specific enzyme inhibitors elicit a differential response in temperature-sensitive structural variants of this enzyme and whether, in principle, structural variants of virus enzymes or the respective ts- mutants in vivo are suitable as a screening system for selective antiviral agents.

DNA-Directed DNA Polymerase↗
PubMed2003

Genetic mutation associated with meiotic metaphase-specific apoptosis in MRL/MpJ mice.

It has been reported that the MRL/MpJ mouse strain shows several unique phenotypes, including rapid wound healing, inherent collagen disease, heat shock-resistant spermatocytes, and metaphase-specific apoptosis (Msa) in the testis. In the present study, we found the genetic mutation associated with Msa by chromosomal mapping with 555 backcross progeny. The Sertoli cell index of abnormal metaphasic spermatocytes was clearly divided into two groups in the first 200 male backcross progeny, which were created by mating female F1 (female C57BL/6 x male MRL/MpJ) with male MRL/MpJ mice, indicating that Msa was caused by only one gene. The result of chromosomal mapping throughout the 555 backcross progeny by using microsatellite markers and single nucleotide polymorphism (SNP) revealed that Msa was mapped on the telomeric region of chromosome 1 and was significantly linked with exonuclease 1 (Exo1) and choroideremia-like (rab escort protein 2) (Chml/Rep2) genes. It was found that the Chml/Rep2 gene was not a candidate for Msa by means of the nucleotide sequences of several inbred strains. On the Exo1 gene in strain MRL/MpJ, but not in other strains, it was surprisingly noted that the truncated forms (tr1-Exo1 and tr2-Exo1) were expressed in all tissues examined as well as normal Exo1 by reverse transcriptase-polymerase chain reaction (RT-PCR). Additionally, the truncated forms of the Exo1 gene were suggested to be transcribed by alternative splicing of the 9th exon, possibly resulting from nucleotide substitution of the branch site existing in the 8th intron. These results suggested that the testicular meiotic Msa in MRL/MpJ mice was a unique phenotype caused by incomplete alternative splicing of the Exo1 gene.

Animals↗
PubMed2003

Enzymatic strategies for the characterization of nucleic acids by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) is a powerful technique used for the identification and characterization of DNA polymorphisms. Continual improvement in instrument design assures high mass measurement accuracy, sensitivity, and resolving power. This work describes an eclectic array of enzymatic strategies we have invoked in order to detect single-nucleotide polymorphisms by ESI-MS, although other applications may be envisioned. One strategy combines the use of two enzymes, exonuclease III and lambda exonuclease, to provide a ladder of single-stranded DNA fragments for straightforward sequence identification by mass spectrometry. A second strategy combines restriction enzymes to screen for polymorphisms present within specific amplicons. Finally, we describe the use of stable-isotope-labeled nucleotides for the determination of length and base composition of a PCR product.

Exodeoxyribonucleases↗
PubMed1993

On the activities of Escherichia coli exonuclease III.

Exonuclease III (Exo III) of Escherichia coli is a DNA-modifying enzyme very frequently used in molecular biology. The experiments described here were carried out with the aims of reliably controlling exonuclease activity and of learning more about the enzyme's specificities. The dependence of Exo III activity on factors such as temperature (including heat inactivation), the concentrations of Exo III and NaCl, and the concentration and shape of 3' termini was investigated. Double stranded DNA was found to be a competitive inhibitor of the enzyme activity. Some four nucleotide 3' protrusions were shown to be sensitive to Exo III digestion. The synchronism of deletion was also examined. Implications for the proposed mechanism of activity are discussed.

Base Sequence↗
PubMed1994

Homogeneous fluorescence detection method for human leukocyte antigen-DR typing following polymerase chain reaction amplification with sequence-specific primer.

A fluorescent homogeneous method for the detection of sequence-specific amplification of human leukocyte antigen (HLA) alleles has been developed. In this approach, polymerase chain reaction sequence-specific primers (PCR-SSP) are used to amplify DRB1, DRB3, and DRB4 alleles. Lambda exonuclease and Exonuclease I are added to reduce background by digesting template DNA, partial primer dimer, and primer. PCR amplicons are then detected following the addition of a fluorescent dye (thiazole yellow dimer) which binds to double-stranded DNA. No transfer or wash steps are required. Thus, the risk of sample contamination, which is a major source of inaccuracy for DNA amplification methods, is greatly reduced. This approach is also faster and more easily automated than the standard approach using gel electrophoresis and ethidium bromide staining. Speed and automation are important considerations for HLA typing since the number of possible alleles for each HLA type is substantial. A homogeneous HLA-SSP typing method may be especially useful for clinical labs doing large numbers of samples and for the eventual automation of HLA DNA typing.

Alleles↗
PubMed1995

DNA probe assay based on exonuclease III digestion of probes hybridized on target DNA.

A quantitative DNA probe assay process has been developed that uses exonuclease III. The fluorophore-labeled DNA probe is hybridized with specific sequences of the target DNA and then enzymatically digested. As these probe hybridization and digestion cycle reactions are repeated at a fixed temperature, digested probes (shortened probes) accumulate in the reaction mixture in a manner similar to a DNA polymerase chain reaction. Investigation of the digestion characteristics of the DNA probe showed that a slight digestion of a free single-stranded probe produces a large background signal, which results in low detection sensitivity. The digestion of single-stranded DNA probes is caused by double-stranded formations in the molecules. This digestion decreases and the double-stranded-specific digestion increases with increasing reaction temperature. When the reaction occurs at 45 degrees C, the association rate of the enzyme on the double-stranded DNA is 700 times faster than that on single-stranded DNA. This enables selective digestion of double-stranded DNA. The detection limit is 9 x 10(-19) mol for a M13-phage DNA.

Animals↗
PubMed1998

Adaptor ligation-based polymerase chain reaction-mediated walking.

An improved method of adaptor ligation PCR was developed for isolation of unknown sequences flanking a known DNA sequence. It was determined that the specificity of the adaptor ligation-based walking technique could be significantly enhanced by using uniquely blocked adaptors along with removal of unligated genomic DNA by exonuclease III digestion. This technique was utilized to isolate three novel promoter regions from three different Zea mays (maize) peroxidase genes. Sequences encoding a putative maize 6-phosphogluconate dehydrogenase gene were also isolated and confirmed by sequence analysis. The described improvements could be applied to other existing adaptor ligation-based PCR walking techniques.

Base Sequence↗
PubMed2000

Method enabling pyrosequencing on double-stranded DNA.

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

Alkaline Phosphatase↗
PubMed2001

Lambda exonuclease-based subtractive hybridization approach to isolate differentially expressed genes from leaf cultures of Paulownia kawakamii.

Genes that are preferentially expressed in a particular developmental pathway can be isolated by subtractive hybridization (SH). We developed a PCR-based approach coupled with lambda exonuclease digestion that allows for generating single-stranded tester and driver nucleic acids suitable for SH starting from cDNA libraries. An efficient subtraction strategy was developed to overcome some of the problems in the previously described SH protocols, such as the need for large amounts of experimental tissue, RNase contamination during solution hybridization, and postsubtraction recovery of nucleic acids. We used this method to obtain cDNA corresponding to genes expressed during adventitious shoot regeneration from excised leaf cultures of the fast-growing tree Paulownia kawakamii. Over 36 cDNA clones were isolated and 1 of the differentially expressed clones codes for a leucine zipper transcription factor. This clone showed about sixfold higher level of expression in the shoot-forming tissues (tester) compared to that in the callus-forming tissues (driver) of Paulownia, suggesting that differentially expressed genes can be efficiently isolated using this simple lambda exonuclease-based subtractive hybridization method.

Amino Acid Sequence↗
PubMed1993

DNA binding and cleavage of a novel antitumor antibiotic dynemicin A: detection of binding and cleavage sites by exonuclease III digestion and alkali-induced cutting reactions.

We found here that dynemicin A effectively breaks DNA strands under alkaline pH condition. Binding of dynemicin A to double-stranded DNA clearly interrupts the digestion reaction of exonuclease III. The DNA association sites of dynemicin A correspond considerably well to its cleavage sites. Dynemicin A seems to intercalate preferentially into the relaxed region of the DNA double helix. On the other hand, the alkali-product of dynemicin A was chromatographically identified with dynemicin N, suggesting a DNA cleavage mechanism similar to the reductant- and light-induced activation systems of dynemicin A. In order to detect drug binding to the relaxation structure in the DNA duplex, the present exonuclease III-digestion-stop-sequence method is useful.

Anthraquinones↗
PubMed1993

Sensitive detection of low levels of ribonuclease H activity by an improved renaturation gel assay.

Renaturation gel assays are good tools to assign enzymatic activities to protein bands. First, proteins are separated by denaturating electrophoresis on substrate-containing gels. Then, following the elimination of the denaturing agent, polypeptides are allowed to renature, thus leading to the degradation of the embedded substrate at positions at which the corresponding activity has moved. Nevertheless, this in situ technique does not only reflect a certain amount of enzyme activity, it also depends upon the ability of an enzyme to renature. Here we present a renaturation gel assay procedure with an improved sensitivity and discuss the detection of E. coli and human ribonuclease H activities as an example.

DNA Polymerase I↗
PubMed1997

Reversible inhibition of lambda exonuclease with high pressure.

The hydrolytic activity of lambda exonuclease, a highly processive enzyme, is inhibited by high pressure. When assayed at 67 MPa, the activity is 87% of the atmospheric rate; increasing the pressure to 336 MPa causes a greater than 99% inhibition of the enzyme activity. Decreasing the pressure from 336 MPa to 67 MPa at 20 degrees C reverses the inhibition. The use of hydrostatic pressure to control the activity of lambda exonuclease has potential practical application in DNA sample preparation, analysis, and sequencing.

Atmospheric Pressure↗
PubMed1997

Formation of novel C1-oxidised abasic sites in alkylperoxyl radical-damaged plasmid DNA.

We have recently shown that peroxyl radicals react with DNA to form alkali-labile sites. To further characterise these lesions, we studied their susceptibility to digestion by repair endonucleases that recognise different types of abasic sites. We found that peroxyl radical-damaged pSP189 plasmids were resistant to cleavage by T4 endonuclease V, an enzyme that incises DNA at "regular" and C4-oxidised abasic residues. In contrast, the DNA was digested by exonuclease III, an enzyme that recognises "regular" and C1-oxidised abasic sites. The presence of Trolox during exposure to peroxyl radicals reduced subsequent DNA cleavage by exonuclease III, while prior incubation of damaged plasmids with methoxyamine potentiated digestion by this enzyme. These findings suggest that peroxyl radical-induced DNA damage involves the generation of novel C1-oxidised deoxyribose residues.

Amidines↗
PubMed1999

Recognition of sequence-directed structure of the ssDNA backbone by nucleases.

Escherichia coli endonuclease I and exonuclease VII appear to recognize sequence-dependent conformations in the ssDNA backbone. ssDNAs, containing either A- and/or T-tract or a CAP binding region, were digested with these nucleases under conditions which minimize the formation of secondary structures. The digestion patterns were examined in relation to previous results of biochemical and crystallographic studies on dsDNA, and showed broad agreement. Endonuclease I cleaved ssDNA at sites corresponding to bent sites in dsDNA.

Bacterial Proteins↗
PubMed2001

Preferential expression of an intact WRN gene in Werner syndrome cell lines in which a normal chromosome 8 has been introduced.

Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WRN gene. All mutations of the WRN gene reported thus far are predicted to produce the truncated WRN proteins. The mRNAs that contain chain-termination mutations are supposed to be unstable due to degradation by nonsense-mediated mRNA decay (NMD). In the present study, we investigated the expressions of intact and nonsense-mutated WRN genes in Werner syndrome cell lines in which a normal chromosome 8 had been introduced by microcell fusion. We demonstrate here that the expression of the mutated WRN gene that produces nonsense mRNAs remains at low levels, resulting in the preferential expression of the intact WRN gene in the WS microcell hybrids. This result supports the idea that imperfect messages containing premature termination codons are eliminated by the RNA surveillance system, suggesting the significance of the NMD mechanism in the etiology of Werner syndrome.

Cell Line↗
PubMed2002

Analysis of the role of RecQ helicases in RNAi in mammals.

The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm, and RecQ1 genes that are related to Mut-7 and Qde3, two genes essential for RNAi in Caenorhabditis elegans and quelling in Neurospora, respectively. Our results suggest that Wrn, Blm, and RecQ1 are not involved in sequence-specific mRNA degradation in mammals in response to dsRNA, suggesting potential differences in the mammalian RNAi pathway.

Adenosine Triphosphatases↗
PubMed1998

Werner helicase is localized to transcriptionally active nucleoli of cycling cells.

Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS), a "caricature of aging." We have localized the Werner protein (WRNp) to the nucleoli of replicating mammalian cells, where its appearance is associated with transcriptional activity. A dramatic reduction of the nucleolar signal and of [3H]uridine incorporation occurred when cultures were made quiescent or were exposed to 4-nitroquinoline-1-oxide (4NQO), to which WS cells are particularly susceptible. Total cellular levels of WRNp, however, did not change, and virtually all WRNp was in the nuclear fractions, consistent with translocation to the nucleoplasm and/or masking of the epitopes. The 4NQO-induced altered state of WRNp was prevented by Na3VO4, but not by okadaic acid, suggesting that WRNp localization/function is partially regulated by kinases/phosphatases for Tyr substrates on WRNp or interacting proteins. The repression of rDNA transcription by 4NQO was not reversed by Na3VO4. We suggest that physiological states and genotoxic agents modulate the interaction of WRNp with rDNA, consistent with a role of WRNp in rDNA transcription.

4-Nitroquinoline-1-oxide↗
PubMed1995

Isolation and characterization of the human MRE11 homologue.

Mutation of the Saccharomyces cerevisiae RAD52 epistasis group gene, MRE11, blocks meiotic recombination, confers profound sensitivity to double-strand break damage, and has a hyperrecombinational phenotype in mitotic cells. We isolated a highly conserved human MRE11 homologue using a two-hybrid screen for DNA ligase I-interacting proteins. Human MRE11 shares approximately 50% identity with its yeast counterpart over the N-terminal half of the protein. MRE11 is expressed at the highest levels in proliferating tissues, but is also observed in other tissues. The MRE11 locus maps to human chromosome 11q21 in a region frequently associated with cancer-related chromosomal abnormalities. A MRE11-related locus was found on chromosome 7q11.2-q11.3.

Amino Acid Sequence↗