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1.
Biotechnol Adv ; 36(3): 603-612, 2018.
Article in English | MEDLINE | ID: mdl-29248681

ABSTRACT

3'-nucleases/nucleotidases of the S1-P1 family (EC 3.1.30.1) are single-strand-specific or non-specific zinc-dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single-strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure-function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.


Subject(s)
Antineoplastic Agents/pharmacology , Biotechnology/methods , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/metabolism , DNA Mutational Analysis/methods , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Host-Pathogen Interactions , Humans , Molecular Targeted Therapy/methods , Nucleotidases/metabolism , Single-Strand Specific DNA and RNA Endonucleases/genetics , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Structure-Activity Relationship , Substrate Specificity
2.
Proc Natl Acad Sci U S A ; 106(31): 12861-6, 2009 Aug 04.
Article in English | MEDLINE | ID: mdl-19628696

ABSTRACT

The unusual structural forms of telomere DNA, which protect the ends of chromosomes during replication, may render it vulnerable to unprecedented photodamage, possibly involving nonadjacent bases that are made proximate by folding. The G-quadruplex for the human telomere sequence consisting of a repeating d(TTAGGG) is one unusual form. Tel22, d[AGGG(TTAGGG)(3)], forms a basket structure in the presence of Na(+) and may form multiple equilibrating structures in the presence of K(+) with hybrid-type structures predominating. UVB irradiation of d[AGGG(TTAGGG)(3)] in the presence of Na(+) results in a cis,syn thymine dimer between two adjacent Ts in a TTA loop and a mixture of nonadjacent anti thymine dimers between various loops. Irradiation in the presence of K(+), however, produces, in addition to these same products, a large amount of specific anti thymine dimers formed between either T in loop 1 and the central T in loop 3. These latter species were not observed in the presence of Na(+). Interloop-specific anti thymine dimers are incompatible with hybrid-type structures, but could arise from a chair or basket-type structure or from triplex intermediates involved in interconverting these structures. If these unique nonadjacent anti thymine dimer photoproducts also form in vivo, they would constitute a previously unrecognized type of DNA photodamage that may interfere with telomere replication and present a unique challenge to DNA repair. Furthermore, these unusual anti photoproducts may be used to establish the presence of G-quadruplex or quadruplex-like structures in vivo.


Subject(s)
G-Quadruplexes , Pyrimidine Dimers/chemistry , Telomere , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Fungal Proteins/pharmacology , Humans , Photochemistry , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Spectrometry, Mass, Electrospray Ionization
3.
Neoplasma ; 53(5): 402-9, 2006.
Article in English | MEDLINE | ID: mdl-17013534

ABSTRACT

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.


Subject(s)
Neoplasms, Experimental/drug therapy , Phaseolus/enzymology , Single-Strand Specific DNA and RNA Endonucleases/immunology , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Animals , Antineoplastic Agents/pharmacology , Antispermatogenic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Embryo, Mammalian/drug effects , Humans , Male , Mice , Mice, Nude , Ribonuclease, Pancreatic/immunology , Ribonuclease, Pancreatic/pharmacology , Spermatogenesis/drug effects , Spermatozoa/drug effects , Teratogens/pharmacology
4.
Biophys Chem ; 112(1): 59-67, 2004 Dec 01.
Article in English | MEDLINE | ID: mdl-15501576

ABSTRACT

Binding of zinc to a 19 mer double-stranded oligodeoxyribonucleotide was investigated by anodic stripping voltammetry and cyclic voltammetry in order to understand the roles of zinc in DNA cleavage catalyzed by mung bean nuclease. These methods rely on the direct monitoring of zinc oxidation current in the absence and in the presence of the oligo. Zinc titration curves with the ds-oligodeoxyribonucleotide were obtained in concentrations ranging from 3.62 x 10(-9) to 3.62 x 10(-8) M and 4.06 x 10(-10) to 5.25 x 10(-9) M. The acquired data were used to determine the dissociation constant, stoichiometry and zinc binding sites of the complex and to understand the specific changes of ds-oligodeoxyribonucleotide secondary structure by zinc binding. The oxidation-reduction process of zinc was also investigated by cyclic voltammetry through I (oxidation current) versus v(1/2) (square root of scan rate) curves in the absence and in the presence of the double-stranded oligodeoxyribonucleotide.


Subject(s)
DNA, Single-Stranded/metabolism , DNA/metabolism , Electrochemistry , Oligodeoxyribonucleotides/metabolism , Zinc/metabolism , Bacteriophage lambda/genetics , Binding Sites , Cations, Divalent , Kinetics , Models, Molecular , Oxidation-Reduction , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Titrimetry , Zinc/chemistry
5.
EMBO J ; 21(24): 6925-34, 2002 Dec 16.
Article in English | MEDLINE | ID: mdl-12486013

ABSTRACT

The incorporation of selenocysteine into proteins is directed by specific UGA codons and mRNA secondary structures, designated SECIS elements. In bacteria, these elements are positioned within the reading frame of selenoprotein mRNAs immediately downstream of the triplet coding for selenocysteine, and they tether a complex of the selenocysteine-specific elongation factor SelB, GTP and selenocysteyl-tRNA(Sec) to the site of UGA decoding. A SECIS-like structure was identified in the 5' non-translated region of the selAB transcript, encoding selenocysteine synthase and SelB. It specifically binds to SelB and the formation of a SelB.GTP.selenocysteyl-tRNA(Sec) complex on the SECIS-like element represses expression of the downstream gene. This effect is abolished by mutations preventing formation of the complex. The regulatory pattern observed correlated with the levels of sel gene products. As quaternary complex formation on the SECIS-like element did not influence the transcription rate and only slightly reduced the level of selAB mRNA, it was concluded that the structure is involved in regulating translation initiation efficiency, thereby coupling selenocysteine biosynthesis to the availability of the trace element selenium.


Subject(s)
Escherichia coli/metabolism , Protein Biosynthesis , RNA/metabolism , Bacterial Proteins/metabolism , Base Sequence , Immunoblotting , Lac Operon , Models, Biological , Models, Genetic , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Selenium/metabolism , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Time Factors , Transcription, Genetic , beta-Galactosidase/metabolism
6.
J Bacteriol ; 184(14): 3984-91, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12081971

ABSTRACT

We found that the biosynthesis of actinorhodin (Act), undecylprodigiosin (Red), and calcium-dependent antibiotic (CDA) are dramatically activated by introducing certain mutations into the rpoB gene that confer resistance to rifampin to Streptomyces lividans 66, which produces less or no antibiotics under normal growth conditions. Activation of Act and/or Red biosynthesis by inducing mutations in the rpoB gene was shown to be dependent on the mutation's position and the amino acid species substituted in the beta-subunit of the RNA polymerase. Mutation analysis identified 15 different kinds of point mutations, which are located in region I, II, or III of the rpoB gene and, in addition, two novel mutations (deletion of nucleotides 1287 to 1289 and a double substitution at nucleotides 1309 and 1310) were also found. Western blot analyses and S1 mapping analyses demonstrated that the expression of actII-ORF4 and redD, which are pathway-specific regulatory genes for Act and Red, respectively, was activated in the mutants able to produce Act and Red. The ActIV-ORF1 protein (an enzyme for Act biosynthesis) and the RedD protein were produced just after the upregulation of ActII-ORF4 and RedZ, respectively. These results indicate that the mutation in the rpoB gene of S. lividans, resulting in the activation of Act and/or Red biosynthesis, functions at the transcription level by activating directly or indirectly the key regulatory genes, actII-ORF4 and redD. We propose that the mutated RNA polymerase may function by mimicking the ppGpp-bound form in activating the onset of secondary metabolism in STREPTOMYCES:


Subject(s)
Anthraquinones/metabolism , Anti-Bacterial Agents/biosynthesis , Mutation , Plant Proteins/genetics , Prodigiosin/biosynthesis , Streptomyces/metabolism , Amino Acid Sequence , Blotting, Western , DNA-Directed RNA Polymerases , Molecular Sequence Data , Multigene Family , Prodigiosin/analogs & derivatives , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Streptomyces/genetics , Transcription, Genetic
7.
DNA Repair (Amst) ; 1(6): 437-47, 2002 Jun 21.
Article in English | MEDLINE | ID: mdl-12509232

ABSTRACT

8-Methyl-2'-deoxyguanosine (8-medGuo) has been shown to be a major stable alkylation product of 2'-deoxyguanosine induced by methyl radical attack on DNA. Moreover, by using primer extension assays, the latter DNA modification has recently been reported to be a miscoding lesion by generating G to C and G to T transversions and deletions in vitro. However, no data have been reported up to now, concerning the processing of this C8-alkylated nucleoside by the DNA repair machinery. Therefore, we have investigated the capability of excision of 8-methylguanine (8-meGua) site specifically incorporated into oligonucleotide substrates by several bacterial, yeast and mammalian DNA N-glycosylases. The results show that the 3-methyladenine (3-meAde) DNA glycosylase II (AlkA protein) from Escherichia coli is the only DNA N-glycosylase tested able to remove 8-meGua from double-stranded DNA fragments. Moreover, the activity of AlkA for 8-meGua varied markedly depending on the opposite base in DNA, being the highest with Adenine and Thymine and the lowest with Cytosine and Guanine. The removal of 8-meGua by AlkA protein was compared to that of 7-methylguanine (7-meGua) and hypoxanthine (Hx). The rank of damage as a substrate for AlkA being 7-meGua>8-meGua>Hx. In contrast, the human 3-meAde DNA N-glycosylase (Mpg) is not able to release 8-meGua paired with any of the four DNA bases. We also show that, DNA N-glycosylases involved in the removal of oxidative damage, such as Fpg or Nth proteins from E. coli, Ntg1, Ntg2 or Ogg1 proteins of Saccharomyces cerevisiae, or human Ogg1 do not release 8-meGua placed opposite any of the four DNA bases. Furthermore, HeLa and Chinese hamster ovary (CHO) cell free protein extracts do not show any cleavage activity at 8-meGua paired with adenine or cytosine, which suggests the absence of base excision repair (BER) of this lesion in mammalian cells.


Subject(s)
DNA Repair/physiology , DNA/metabolism , Escherichia coli/enzymology , Guanine/analogs & derivatives , Guanine/metabolism , N-Glycosyl Hydrolases/physiology , Alkaline Phosphatase/pharmacology , Animals , Base Pair Mismatch , CHO Cells , Chromatography, High Pressure Liquid , Cricetinae , Cytosine/chemistry , DNA Damage/genetics , DNA Glycosylases , DNA Primers , Exonucleases/pharmacology , HeLa Cells , Humans , In Vitro Techniques , N-Glycosyl Hydrolases/genetics , N-Glycosyl Hydrolases/isolation & purification , N-Glycosyl Hydrolases/metabolism , Oligodeoxyribonucleotides/chemical synthesis , Saccharomyces cerevisiae/enzymology , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
8.
Mol Cell ; 6(3): 563-72, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11030336

ABSTRACT

Yeast Rad51 recombinase has only minimal ability to form D loop. Addition of Rad54 renders D loop formation by Rad51 efficient, even when topologically relaxed DNA is used as substrate. Treatment of the nucleoprotein complex of Rad54 and relaxed DNA with topoisomerases reveals dynamic DNA remodeling to generate unconstrained negative and positive supercoils. DNA remodeling requires ATP hydrolysis by Rad54 and is stimulated by Rad51-DNA nucleoprotein complex. A marked sensitivity of DNA undergoing remodeling to P1 nuclease indicates that the negative supercoils produced lead to transient DNA strand separation. Thus, a specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation.


Subject(s)
DNA, Superhelical/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Recombination, Genetic/genetics , Saccharomyces cerevisiae Proteins , Yeasts/genetics , Adenosine Triphosphate/metabolism , DNA Helicases , DNA Repair Enzymes , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Enzymologic , Nucleic Acid Conformation , Rad51 Recombinase , Recombination, Genetic/drug effects , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Yeasts/enzymology
9.
Rapid Commun Mass Spectrom ; 14(1): 26-32, 2000.
Article in English | MEDLINE | ID: mdl-10623924

ABSTRACT

Fluorescein-labeled oligodeoxynucleotides (ODNs) from automated synthesis commonly produce multiple peaks in high performance liquid chromatography (HPLC) chromatograms. We found that these peaks are due to chemical modifications of the ODNs instead of the common perception of isomers. To identify the modifications, a model ODN, fluorescein-T(25), was synthesized and five compounds were isolated. Nuclease P1 (NP1) digestion was employed to cleave these compounds into nucleotides and fluorescein-nucleotides in order that the modifications be determined by mass spectrometry (MS). Analyses of NP1 digestion products containing fluorescein by MS revealed the expected product F1-T (M) and four unexpected compounds with MWs at M-1, M-17, M-16 and M + 16, respectively. Collision-induced dissociation (CID) spectra of these digestion products indicate that all modifications occur on the thiourea linkage [-NH-C( = S)-NH-] to the fluorescein moiety and the adjacent phosphate group, and the modifications were determined. The modifications were also confirmed by accurate mass measurement with Fourier transform mass spectrometry (FT-MS), by the synthesis of a reference compound, and by a mechanistic study using model compounds. These results demonstrate the power of the mass spectrometric techniques by determining the structures of two pairs of ODNs with MW difference of 1 Da. The results also suggest that fluorescein phosphoramidite with a thiourea linkage is not appropriate for the automated synthesis of fluorescein-labeled ODNs of high purity.


Subject(s)
Contrast Media , Fluorescein , Oligodeoxyribonucleotides/chemistry , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Chromatography, High Pressure Liquid , Guanidines/analysis , Molecular Weight , Oligodeoxyribonucleotides/isolation & purification , Oligodeoxyribonucleotides/metabolism , Reference Standards , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/standards , Thiourea/analogs & derivatives , Thiourea/analysis
10.
FEBS Lett ; 440(1-2): 38-40, 1998 Nov 27.
Article in English | MEDLINE | ID: mdl-9862420

ABSTRACT

The complementary-addressed modification of DNA and proteins in chromatin using photoreactive derivatives of pd(AC)6 has been studied. These oligonucleotides form complementary complexes with specific DNA sequences and modify both DNA and proteins in the vicinity of these regions, and can be used for investigation of the protein environment in DNA. We have demonstrated that photoreactive derivatives of oligonucleotides can quickly and efficiently modify chromatin proteins and seem to be promising for investigation of perturbations in chromatin structure during the cell cycle. A comparison between modified chromatin from synchronized cells has demonstrated differences in the sets of proteins modified in the S and G1/S phases of the cell cycle. An increase in spermine and spermidine concentrations leads to an increase in modification of definite chromatin proteins. It can be supposed that the B-Z transition that can be stabilized by the presence of natural polyamines is one of the reasons for the presence of single-stranded DNA regions, containing sets of (dG-dT)n and accessible for interaction with complementary oligonucleotides.


Subject(s)
Chromatin/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Deoxyribonucleotides/metabolism , Photoaffinity Labels/metabolism , Polyamines/pharmacology , Cell Nucleus/metabolism , Chromatin/chemistry , DNA/chemistry , DNA/metabolism , DNA, Single-Stranded/metabolism , Deoxyribonucleotides/chemistry , G1 Phase , HeLa Cells , Humans , Molecular Weight , Nucleic Acid Conformation , Photoaffinity Labels/chemistry , S Phase , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Spermidine/pharmacology , Spermine/pharmacology
11.
J Mol Biol ; 283(1): 43-58, 1998.
Article in English | MEDLINE | ID: mdl-9761672

ABSTRACT

Bacteriophage N4 virion RNA polymerase promoters contain five to seven-base inverted repeats separated by three bases and centered at position -12 from the site of transcription initiation. We have previously shown that these inverted repeats extrude as hairpins at physiological superhelical densities in a Mg(II)-dependent manner. Mg(II)-dependent hairpin extrusion at promoters P1 and P2 displays quantitative differences in reactivity to structural probes at different DNA superhelical densities, with extrusion at P2 being more favored at low superhelical density. Analyses of mutant promoters using structure-specific probes revealed that specific sequences, at the closing base-pair of the hairpin and at the loop (i.e. 5'-C-GXA-G-3' where X=G, A, T), are required for extrusion of the small promoter hairpins at physiological superhelical density. The sequence-dependent requirements for extrusion of the small N4 promoter hairpins may be generally applicable for other such sequences found both in prokaryotic and eukaryotic genomes.


Subject(s)
DNA, Viral/chemistry , DNA-Directed RNA Polymerases , Nucleic Acid Conformation , Podoviridae/genetics , Promoter Regions, Genetic/genetics , Acetaldehyde/analogs & derivatives , Acetaldehyde/pharmacology , Base Sequence , Conserved Sequence , DNA, Superhelical , DNA, Viral/genetics , DNA-Directed RNA Polymerases/metabolism , Mutation , Podoviridae/enzymology , Potassium Permanganate/pharmacology , Repetitive Sequences, Nucleic Acid/genetics , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Virion/enzymology
12.
J Histochem Cytochem ; 46(3): 389-95, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9487121

ABSTRACT

We describe a new technique that allows specific visualization of RNA at the electron microscopic level by means of terbium citrate. Under the conditions presented here, terbium binds selectively to RNA and stains nucleoli, interchromatin granules, peri-chromatin fibrils, perichromatin granules, and coiled bodies in the cell nucleus, whereas ribosomes are the only contrasted structures in the cytoplasm. All the cell components contrasted by terbium are known to contain RNA. When ultrathin sections are pretreated with RNase A or nuclease S1 (specific for single-stranded nucleic acids), staining does not occur. Neither DNase nor pronase influences the reaction. We conclude that terbium staining is selective for RNA and especially for single-stranded RNA. The staining can be performed on thin sections of material embedded both in epoxy and in acrylic resins. The technique is not influenced by the aldehyde fixative used and can also be utilized after immunolabeling. The endproduct is very fine and, although weak in contrast, is suitable for high-resolution observations.


Subject(s)
Histocytochemistry/methods , Microscopy, Electron/methods , RNA/ultrastructure , Terbium , Adenoviridae/isolation & purification , Animals , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , DNA/analysis , Deoxyribonucleases/pharmacology , HeLa Cells , Humans , Immunohistochemistry , Liver/ultrastructure , Male , Mice , Pancreas/ultrastructure , Pronase/pharmacology , RNA/drug effects , Rats , Ribonuclease, Pancreatic/pharmacology , Ribosomes/drug effects , Ribosomes/ultrastructure , Salmon , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Spermatozoa/ultrastructure , Testis/ultrastructure , Tissue Embedding
13.
Mol Microbiol ; 27(1): 187-95, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9466266

ABSTRACT

A novel type of superoxide dismutase containing nickel as a cofactor (NiSOD) has been discovered in several Streptomyces spp. The gene for NiSOD (sodN) was cloned from S. coelicolor Müller using degenerate oligonucleotide probes designed from the N-terminal peptide sequence of the purified enzyme. It encodes a polypeptide of 131 amino acids (14703 Da), without any apparent sequence similarity to other known proteins. The N-terminus of the purified NiSOD was located 14 amino acids downstream from the initiation codon of the deduced open reading frame (ORF), indicating the involvement of protein processing. The molecular mass of the processed polypeptide was predicted to be 13201 Da, in close agreement with that of the purified NiSOD (13.4 kDa). The transcription start site of the sodN gene was determined by S1 mapping and primer extension analysis. Ni2+ regulates the synthesis of NiSOD polypeptide. S1 mapping of both 5' and 3' ends of sodN mRNA revealed that Ni2+ increased the level of monocistronic sodN mRNA by more than ninefold without changing its half-life, thus demonstrating that Ni2+ regulates transcription. Both precursor and processed NiSOD polypeptides with little SOD activity were produced from the cloned sodN gene in S. lividans in the absence of sufficient Ni2+; however, on addition of Ni2+, active NiSOD consisting of only processed polypeptide was formed. Expression of the full-length sodN gene in E. coli produced NiSOD polypeptide without any SOD activity even in the presence of Ni2+. However, deletion of nucleotides encoding the N-terminal 14 amino acids from the sodN gene allowed the production of active NiSOD in E. coli, indicating that N-terminal processing is required to produce active NiSOD. These results reveal the unique role of nickel as a multifaceted regulator in S. coelicolor controlling sodN transcription and protein processing, as well as acting as a catalytic cofactor.


Subject(s)
Gene Expression Regulation, Bacterial/genetics , Gene Expression Regulation, Enzymologic/genetics , Nickel/physiology , Streptomyces/enzymology , Superoxide Dismutase/genetics , Amino Acid Sequence , Amino Acids/analysis , Base Sequence , Blotting, Western , Cloning, Molecular , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Nickel/chemistry , Nucleic Acid Hybridization , Open Reading Frames/genetics , RNA Processing, Post-Transcriptional , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Single-Strand Specific DNA and RNA Endonucleases/chemistry , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Streptomyces/genetics , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , Transcription, Genetic
14.
Biochem Biophys Res Commun ; 238(1): 33-8, 1997 Sep 08.
Article in English | MEDLINE | ID: mdl-9299446

ABSTRACT

The Epstein-Barr virus (EBV) DNA polymerase (Pol) holoenzyme is an essential enzyme required for ori-Lyt dependent EBV DNA replication. Using singly primed M13ssDNA circles as template, the EBV DNA Pol holoenzyme synthesized DNA chains greater than the unit length of M13 ssDNA in addition to full length products even at a low ratio of polymerase molecule per templates. The long replication products consisted of circular double-stranded DNA with single-stranded tails that were sensitive to mung bean nuclease. Reconstitution of the EBV Pol holoenzyme by preincubation of BALF5 Pol catalytic subunit and BMRF1 Pol accessory subunit in vitro resulted in reproduction of the strand displacement DNA synthesis. Thus, the EBV DNA Pol holoenzyme by itself is able to produce strand displacement coupled to the polymerization process in a highly processive way in the absence of any other protein.


Subject(s)
DNA, Viral/biosynthesis , DNA-Binding Proteins , DNA-Directed DNA Polymerase/metabolism , Herpesvirus 4, Human/enzymology , Herpesvirus 4, Human/genetics , Viral Proteins , Adenosine Triphosphate/metabolism , Antigens, Viral/metabolism , Catalysis , DNA Replication/drug effects , DNA, Circular/metabolism , DNA, Single-Stranded/metabolism , DNA, Viral/drug effects , DNA, Viral/metabolism , Humans , Hydrolysis , Single-Strand Specific DNA and RNA Endonucleases/pharmacology
15.
Jpn J Cancer Res ; 88(10): 921-4, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9414650

ABSTRACT

The issue of toxins produced by Helicobacter pylori (H. pylori) urgently requires clarification given that the bacterium causes gastric epithelial cell damage which may lead to precancerous and cancerous changes. During an investigation of the possibility of mono(adenosine 5'-diphosphate (ADP)-ribosyl)ation by H. pylori products, as observed for other bacterial toxins, we found that radioactivity of [adenylate-32P]nicotinamide adenine dinucleotide (NAD) is incorporated into an H. pylori protein of 80 kDa after incubation with crude bacterial extract. In contrast, [carbonyl-14C]NAD did not show any radioactivity incorporation. Unexpectedly, treatment of the modified protein with 0.1 N HCl, but not 0.1 N NaOH, released the AMP moiety. Such chemical properties are characteristic of bacterial DNA ligase-AMP complexes. We found that an antibody raised against Escherichia coli DNA ligase [EC 6.5.1.2] immunoprecipitated the modified 80 kDa protein. Our results indicate that incorporation of radioactivity derived from NAD into the 80 kDa protein was due to adenylation, but not mono(ADP-ribosyl)ation, of the DNA ligase of H. pylori.


Subject(s)
Adenosine Diphosphate Ribose/metabolism , DNA Ligases/metabolism , Helicobacter pylori/metabolism , NAD/metabolism , Nucleoside Diphosphate Sugars/metabolism , Ribonucleosides/analysis , Bacterial Proteins/immunology , DNA Ligases/immunology , Electrophoresis, Polyacrylamide Gel , Helicobacter pylori/isolation & purification , Hydrochloric Acid/pharmacology , Precipitin Tests , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Sodium Hydroxide/pharmacology , Thermolysin/pharmacology
16.
J Biol Chem ; 271(30): 18061-7, 1996 Jul 26.
Article in English | MEDLINE | ID: mdl-8663482

ABSTRACT

Similar imperfect purine/pyrimidine mirror repeat (PMR) elements have previously been identified upstream of the human MUC1 mucin and CFTR genes. These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH. We now present a detailed characterization of the non-B DNA structure responsible for S1 nuclease sensitivity upstream of the MUC1 gene. A approximately 90-base pair (bp) DNA fragment containing a 32-bp PMR element termed M-PMR3 was subcloned into a recombinant vector. This fragment conferred S1 nuclease sensitivity on the resulting supercoiled plasmid. High resolution mapping of sites reactive to S1 and P1 nucleases demonstrates that cleavage occurs within the M-PMR3 element. High resolution mapping with chemical agents selective for non-B DNA provides evidence that M-PMR3 adopts an H-DNA structure (intramolecular triple helix) in the less common H-y5 isomer at low pH. This result is observed in the presence or absence of Mg2+. Mutation of the native M-PMR3 element to create perfect homopurine/homopyrimidine mirror symmetry alters the preferred folding to the more common H-y3 triplex DNA isomer. These results demonstrate that imperfections in mirror symmetry can alter the relative stabilities of different H-DNA isomers.


Subject(s)
DNA/chemistry , Mucin-1/genetics , Mucins/genetics , Nucleic Acid Conformation , Promoter Regions, Genetic , Base Sequence , Cations, Divalent/pharmacology , DNA/drug effects , Humans , Magnesium/pharmacology , Molecular Probes , Molecular Sequence Data , Nucleotides/chemistry , Single-Strand Specific DNA and RNA Endonucleases/pharmacology
18.
Cancer Chemother Pharmacol ; 38(1): 71-80, 1996.
Article in English | MEDLINE | ID: mdl-8603455

ABSTRACT

The sensitivity for DNA adduct formation by antitumor alkylating agents (mechlorethamine, cisplatin and adozelesin) of the postlabeling technique and thin-layer chromatography was studied. Three DNAs were used: a double-stranded 20-bp oligonucleotide of defined sequence, calf thymus DNA and murine leukemia L1210 cellular DNA. With high concentrations of mechlorethamine, there was a marked decrease in normal dGp, a lesser decrease in dAp and dCp and no change in dTp. Using 2D mapping PEI-cellulose thin-layer chromatography analyses, it was found that six mechlorethamine: DNA adducts were produced after a short exposure to mechlorethamine. After an extended time at relatively high drug concentrations there was an alteration in the mechlorethamine: DNA adduct pattern that may reflect the conversion of monoadducts to crosslinked adducts. Similar observations were made with cisplatin and adozelesin. When murine leukemia L1210 cells were treated with 50 microM mechlorethamine or 50 microM cisplatin for 1 h, six or more mechlorethamine: DNA adducts and five cisplatin: DNA adducts were detected. After allowing 6 h. for repair of potentially lethal damage, several adducts were no longer detectable and others appeared with diminished intensity. Nuclease P(1) dephosphorylates normal nucleotides at relatively low enzyme concentrations with variation depending upon the nucleotide. In general, considerably lower concentrations of nuclease P1 were required to dephosphorylate the normal nucleotides than to dephosphorylate the antitumor alkylating agent: nucleotide adducts, thus allowing increased sensitivity of the postlabeling assay. The sensitivity of detection of antitumor alkylating agent: DNA adducts in DNA from treated L1210 cells approached one adduct per 10(7)-10(8) nucleotides. These results suggest that the postlabeling technique may be sufficiently sensitive and specific for the study of the clinically effective levels of antitumor alkylating agents.


Subject(s)
Antineoplastic Agents, Alkylating/metabolism , DNA Adducts/metabolism , Indoles , Animals , Antineoplastic Agents, Alkylating/chemistry , Antineoplastic Agents, Alkylating/toxicity , Autoradiography , Base Sequence , Benzofurans , Chromatography, Thin Layer , Cisplatin/chemistry , Cisplatin/metabolism , Cisplatin/toxicity , Cyclohexanecarboxylic Acids/chemistry , Cyclohexanecarboxylic Acids/metabolism , Cyclohexanecarboxylic Acids/toxicity , Cyclohexenes , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Adducts/analysis , DNA Adducts/genetics , DNA Damage/genetics , Duocarmycins , Isotope Labeling , Leukemia L1210/genetics , Leukemia L1210/pathology , Mechlorethamine/chemistry , Mechlorethamine/metabolism , Mechlorethamine/toxicity , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , Single-Strand Specific DNA and RNA Endonucleases/metabolism , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Thymus Gland/metabolism , Tumor Cells, Cultured
19.
Carcinogenesis ; 17(1): 109-14, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8565118

ABSTRACT

Previous studies reported that metabolic activation of certain polychlorinated biphenyls (PCBs) resulted in binding to protein, RNA and DNA fractions. However, the formation of DNA adducts has not been demonstrated nor have methods been optimized for the detection of such adducts. In the present study we investigated activation and binding to DNA of lower chlorinated biphenyls using 32P-postlabeling. The incubation of 2-chloro-, 3-chloro-, 3,4-dichloro- and 3,4,5-trichlorobiphenyl with calf thymus DNA and liver microsomes from rats treated with phenobarbital and 3-methylcholanthrene, followed by oxidation with a peroxidase, produced 1-3 major adducts. Reaction of deoxyguanosine 3'-monophosphate with metabolites of the congeneric chlorinated biphenyls produced adducts with similar chromatographic mobility as those with DNA, suggesting that guanine was the preferential site of attack. Furthermore butanol and nuclease P1 enrichments showed different adduct recoveries, depending upon the the chlorobiphenyl. Adducts derived from incubations with monochlorobiphenyls were recovered 2- to 3-fold higher with butanol, while the recovery of di- and tri-chlorobiphenyl adducts was 5- to 7-fold higher with nuclease P1. DNA adducts formed during the metabolism of 3,4-dichlorobiphenyl were reduced by the sulfur nucleophiles, glutathione and N-acetyl-L-cysteine, suggesting that reactive semiquinone(s) or quinone(s) are involved. In contrast, the addition of superoxide dismutase increased adduct formation, suggesting that the quinone metabolites are responsible for the major adducts formed. Our results are consistent with the hypothesis that lower chlorinated biphenyls are metabolically activated to electrophilic quinoid species which bind to DNA.


Subject(s)
DNA Adducts/analysis , DNA/metabolism , Polychlorinated Biphenyls/metabolism , Animals , Biotransformation , Male , Polychlorinated Biphenyls/toxicity , Rats , Rats, Sprague-Dawley , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Superoxide Dismutase/pharmacology
20.
Am J Respir Cell Mol Biol ; 13(6): 703-11, 1995 Dec.
Article in English | MEDLINE | ID: mdl-7576708

ABSTRACT

We have found that preparations of DNA isolated from purulent sputum possess a novel activity which accelerates and stabilizes the binding of human leukocyte elastase to secretory leukoprotease inhibitor, a major endogenous antielastase in the respiratory tract. DNA in sputum is derived from the nuclear debris of disintegrated inflammatory leukocytes, and can attain concentrations ranging from 10(2) to 10(4) micrograms/ml, depending on the severity of pulmonary infection and inflammation. In the presence of 23 micrograms/ml DNA, a concentration lower than those found in most purulent sputa, the rate constant for association of secretory leukoprotease inhibitor with elastase is increased to 1.1 x 10(8) M-1s-1, 44-fold greater than that in the absence of DNA. The equilibrium dissociation constant for the enzyme-inhibitor complex drops to 0.7 pM, two orders of magnitude lower than that in the absence of DNA. The accelerating effect of DNA is further increased by thermal denaturation or by modification with exonuclease III, while it is significantly reduced by digestion with S1 nuclease or by binding of Escherichia coli single-stranded DNA binding protein. The results from these experiments indicate that the structural elements in sputum DNA that are responsible for the accelerating effect have the characteristics of single-stranded sites. Similar kinetic effects on elastase inhibition were also observed with human placental DNA and genomic DNAs from a variety of other species. These findings suggest that DNA in pulmonary secretions may participate in antielastase defense by promoting the binding of secretory leukoprotease inhibitor to leukocyte elastase. The results may have important implications for use of nuclease preparations in mucolytic therapy for cystic fibrosis.


Subject(s)
DNA, Single-Stranded/metabolism , Leukocyte Elastase/metabolism , Mucus/metabolism , Pancreatic Elastase/metabolism , Proteins/metabolism , Serine Proteinase Inhibitors/metabolism , Binding Sites/physiology , Binding, Competitive , Bronchi/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli/chemistry , Exodeoxyribonucleases/pharmacology , Humans , Kinetics , Leukocytes/metabolism , Mucous Membrane/metabolism , Proteinase Inhibitory Proteins, Secretory , Single-Strand Specific DNA and RNA Endonucleases/pharmacology , Sputum/metabolism , Temperature , Trachea/metabolism
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