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PubMed1986

Chromatin condensation and terminal differentiation process in embryonic chicken lens in vivo and in vitro.

During embryonic chick lens differentiation, the epithelial cells become transformed into elongated fibres. Concomitantly, the fibre nuclei undergo degeneration and high molecular weight (HMW) DNA breaks down due to nuclear endodeoxyribonuclease activity. An electronmicroscopic study of lens epithelial and fibre nuclei was made at different stages of chick embryonic development, both in vivo and in vitro. The in vitro conditions are conducive to the expression of endogenous endodeoxyribonuclease activity in fibres. In both conditions we observed condensation of chromatin. The organization of some nuclear material into distinct linear arrays followed by streaming of nuclear material into the cytoplasm is recorded only in vitro. Such a condition may lead to acceleration of the process of aging in lens fibres.

Animals↗
PubMed1972

Virus-associated nucleases: location and properties of deoxyribonucleases and ribonucleases in purified frog virus 3.

At least three nuclease activities are associated with purified frog virus 3. These activities are endodeoxyribonuclease (pH 7.5, double-stranded [DS] and single-stranded [SS] deoxyribonucleic acid [DNA]); endodeoxyribonuclease (pH 5.0, DS and SS DNA); endoribonuclease (DS and SS ribonucleic acid [RNA], pH 7.5). These activities are not adsorbed to the surface of the virion but are within the viral capsid and require detergent disruption of virions to unmask enzyme activity. Only one activity, deoxyribonuclease (pH 5.0, SS and DS DNA) appears to be core-associated after detergent disruption of virions. The ribonuclease degrades poliovirus replicative-form RNA, reovirus native RNA, and poly(I) poly(C) to a product with a sedimentation coefficient of about 6S. Qbeta 6S DS RNA and 4S transfer RNA are not degraded. The ribonuclease appears to be a late function of the virus and is elicited in a soluble form as well as a virus-associated form.

Animals↗
PubMed1969

Bacteriophage-induced inhibition of host functions. II. Evidence for multiple, sequential bacteriophage-induced deoxyribonucleases responsible for degradation of cellular deoxyribonucleic acid.

Degradation of bacterial deoxyribonucleic acid (DNA) after infection with T4 bacteriophage was studied in an endonuclease I-deficient host. The kinetics of degradation were similar to those seen in other hosts with a normal level of this enzyme. Irradiation of extracellular phage with ultraviolet (UV) destroyed the capacity of the infecting virus to induce extensive breakdown of host DNA, which was, however, converted to high-molecular-weight material. Addition of chloramphenicol to T4-infected cells provided data which can be interpreted to indicate the involvement of at least two endodeoxyribonucleases and one exodeoxyribonuclease having a high degree of specificity. A model is proposed showing the sequential action of two endodeoxyribonucleases followed by an exodeoxyribonuclease in the degradation of host DNA. The appearance of these hydrolytic enzymes requires protein synthesis. Infections leading to partial degradation only (UV-irradiated phages, gene 46 mutants) effectively inhibited the synthesis of bacterial messenger ribonucleic acid and of beta-galactosidase.

Chloramphenicol↗
PubMed1987

Formation, detection and repair of AP sites.

The paper is an outline review of the main aspects concerning the formation and repair of AP (apurinic/apyrimidinic) sites in DNA as well as some of the chemical properties allowing their quantitative determination. A new method for the measurement of AP sites based on their reaction with [14C]methoxyamine is described. It has been applied to the measurement of AP sites produced in DNA either by physical (gamma-rays) or chemical (methyl methanesulphonate, osmium tetroxide) agents. The method has also been used to quantify the excision of abnormal bases from DNA under the action of specific DNA glycosylases and to prevent the chemical or enzymatic degradation of DNA containing AP sites. The paper contains data about the purification and characterization of uracil-DNA glycosylase and AP endodeoxyribonuclease from carrot cells, two enzymes involved in the first steps of base excision repair through AP site intermediates. The biological effects of unrepaired AP sites are also discussed.

DNA↗
PubMed1987

Reparative strand incision in saponin-permeabilized human fibroblasts.

The damage-directed strand incision step in the nucleotidyl DNA excision-repair pathway (NDERP) was characterized in quiescent monolayer cultures of human fibroblasts in which the plasma membrane was selectively permeabilized with saponin. When permeable normal human fibroblasts (NHF) were incubated in a DNA-repair assay mixture lacking the deoxyribonucleoside triphosphate precursors, the numbers of UV-dependent DNA-strand breaks were increased by about 9-fold consistent with the uncoupling of incision from gap-filling DNA synthesis and ligation. In uncoupled NHF omission of ATP reduced the numbers of UV-dependent strand breaks by 84% confirming the requirement for ATP for reparative strand incision. Time-course experiments indicated that the maximum rate of strand incision occurred in the first 10 min of incubation of permeable cells and diminished to 16-28% of this rate between 30 and 60 min of incubation. The initial rate of incision in permeable NHF was estimated to be 20% of that seen in intact fibroblasts. Dose-response studies indicated an initial saturation of strand incision activity at fluences between 10 and 25 J/m2. In permeable group A xeroderma pigmentosum fibroblasts (XPA) few UV-dependent incisions were produced after 10-25 J/m2. In the xeroderma pigmentosum variant (XPV) strain that we studied, strand incisions saturated at a plateau level that was about twice that seen in the NHF strain suggesting the preservation of a higher level of incision activity after permeabilization. After fluences above 50 J/m2 additional strand incision was observed in all cell strains reflecting the activity of a damage-dependent endodeoxyribonuclease that is independent of the NDERP. Saponin-treated fibroblasts were also permeable to pancreatic deoxyribonuclease I and the UV-DNA endonuclease from M. luteus indicating that these preparations may be used for in vitro complementation.

Cell Membrane Permeability↗
PubMed1998

Trisomy 21 and accelerated aging: DNA-repair parameters in peripheral lymphocytes of Down's syndrome patients.

Down's syndrome (DS) cases from 1-40 years of age and showing no other anomalies or deficiencies were categorized into three age groups: group 1, < or = 12 years; group 2, 13-25 years; and group 3, > or = 26 years. The DNA-repair markers like unscheduled DNA synthesis (UDS), activities of DNA polymerases, (Total, beta and epsilon) and two endodeoxyribonucleases, (UV- and AP-DNases) were assessed in the peripheral lymphocytes of these subjects (under different conditions) along with age and sex matched normal healthy human subjects. The DS group showed lower DNA-repair efficiency and also an accelerated decline in DNA-repair capacity with age. These results indicate that deteriorated DNA-repair potential could be one of the probable reasons for premature aging seen in this chromosomal disorder.

Adolescent↗
PubMed1987

Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4.

The UV endonucleases [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. We have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV- (4 kJ/m2, 254 nm) treated, [3H]thymine-labeled poly(dA).poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical- (5 kJ/m2, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. Our data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. Our results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.

DNA Glycosylases↗
PubMed1989

Mechanism of DNA cleavage and substrate recognition by a bovine apurinic endonuclease.

The location of the phosphodiester bond cleaved by homogeneous Mg2+-dependent apurinic endodeoxyribonuclease (EC 3.1.25.2; APE) of bovine calf thymus has been determined by using a 21-mer oligonucleotide containing a single central apurinic site as a substrate. A single product of cleavage consistent with cleavage of the oligonucleotide 5' to the apurinic site, and leaving a 3' hydroxyl group, was identified. This enzyme is, therefore, a class II apurinic endonuclease. The substrate specificities of this enzyme have been determined by using a variety of natural and synthetic DNAs or oligonucleotides containing base-free sites. Calf thymus APE has an absolute requirement for a double-stranded DNA and requires an abasic site as a substrate. The presence of a base fragment such as a urea residue, an alkoxyamine group attached to the C'-1 position of the abasic site, or reduction of the C'-1 aldehyde abolishes the APE activity of this enzyme. Synthetic abasic sites containing either ethylene glycol, propanediol, or tetrahydrofuran interphosphate linkages are excellent substrates for bovine APE. These results indicate that APE has no absolute requirement for either ring-opened or ring-closed deoxyribose moieties in its recognition of DNA-cleavage substrates. The enzyme may interact with the pocket in duplex DNA that results from the base loss or with the altered conformations of the phosphodiester backbone that result from the abasic site.

Amines↗
PubMed1995

A novel exocytoplasmic endonuclease from Streptomyces antibioticus.

A new exocytoplasmic, nutritionally controlled endodeoxyribonuclease (EC 3.1.21.-) was purified to homogeneity from Streptomyces antibioticus. The enzyme showed an apparent molecular mass of 29 kDa (being active in the monomeric form) and a pI of approximately 7.8. The nuclease hydrolysed endonucleolytically double-stranded circular and linear DNA. The enzyme makes nicks in one strand of the DNA in G-rich regions, leaving either 5' or 3' short, single-stranded overhangs with 3'-hydroxy and 5'-phosphate termini. Breaks in the DNA occur when two nicks in opposite strands are close together. The enzyme had an optimum pH of 7.5 and an absolute requirement for bivalent cations and > or = 100 mM NaCl in the reaction buffer. Activity was greatly diminished in the presence of phosphate, Hg2+ or iodoacetate and was stimulated by dimethyl sulphoxide. Single-stranded DNA was a much poorer substrate than double-stranded DNA. The nuclease hydrolyses sequences of three or preferably more (dG).(dC) tracts in the DNA. The initial specificity shifts to other sequences (including sequences shorter than those initially hydrolysed) during the course of the reaction, giving the changing pattern of bands observed in agarose gels. 5-Methylcytosine-hemimethylated DNA is not hydrolysed by the nuclease. The properties of this novel enzyme suggest a relationship with class II restriction endonucleases and also with some eukaryotic nucleases.

Base Sequence↗
PubMed1992

Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V.

T4 endonuclease V [endodeoxyribonuclease (pyrimidine dimer); deoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] initiates repair of damaged DNA by hydrolysis of the N-glycosyl bond at the 5' side of a pyrimidine photodimer in double-stranded DNA. To study one of the active sites of T4 endonuclease V, systematic site-directed mutagenesis was performed on the synthetic T4 endonuclease V gene, in parallel with three-dimensional structure analysis by x-ray crystallography. The mutant proteins were evaluated for DNA glycosylase activity using an oligonucleotide duplex (14-mer) containing a single thymidine dimer as a substrate. Replacement of either Glu-23 with glutamine or asparatic acid or Arg-3 with glutamine completely abolished DNA glycosylase activity. Mutation of Arg-3 to lysine or of Arg-26 to glutamine or lysine in a basic amino acid cluster caused serious defects in DNA glycosylase activity, which are reflected in the increases in Km and decreases in kcat of DNA glycosylase activity. On the other hand, substitutions of lysine for Arg-22 or of glutamine for Arg-117 or Lys-121 resulted in increases in the Km value. The completely inactive mutant proteins, E23Q and R3Q, in which glutamine was substituted for Glu-23 and Arg-3, respectively, were further investigated by CD spectroscopy for their ability to bind the oligonucleotide substrate. It was found that the E23Q protein retained specific substrate-binding ability, whereas the R3Q protein did not. These results indicate that Glu-23 plays an important role in catalysis of the DNA glycosylase reaction, and that Arg-3 is a crucial residue for substrate binding. In addition, Arg-22, Arg-26, Arg-117, and Lys-121 in the basic amino acid cluster also participate in substrate binding. We conclude that the basic amino acid cluster in T4 endonuclease V is an essential structure for DNA glycosylase activity.

Amino Acid Sequence↗
PubMed2005

DNase II is a member of the phospholipase D superfamily.

MOTIVATION: DNase II is an endodeoxyribonuclease involved in apoptosis and essential for the mammalian development. Despite the understanding of biochemical properties of this enzyme, its structure and relationships to other protein families remain unknown. RESULTS: Using protein fold-recognition we found that DNase II exhibits a catalytic domain common to the phospholipase D superfamily. Our model explains the available experimental data and provides the first structural platform for sequence-function analyses of this important nuclease.

Amino Acid Sequence↗
PubMed1985

A new approach to the study of the base-excision repair pathway using methoxyamine.

This paper describes the use of methoxyamine to study the enzymatic reactions catalyzed by uracil-DNA glycosylase and by AP (apurinic/apyrimidinic) endodeoxyribonuclease isolated from mammalian cells. [14C]Methoxyamine permits one to follow the formation of AP sites in a uracil-containing polydeoxyribonucleotide incubated with calf thymus uracil-DNA glycosylase. The number of methoxyamine-reacted AP sites is equal to that of uracil released. Methoxyamine has no effect on the uracil-DNA glycosylase activity and may be added together with the enzyme in order to block the AP sites and prevent the degradation of the polynucleotide by the AP endonucleases that may be present in a crude preparation. Addition of methoxyamine to AP sites prevents not only the enzymatic hydrolysis of the adjacent phosphodiester bond but also the degradation of the polynucleotide by NaOH. This protective effect disappears after methoxyamine is removed by acetaldehyde.

Animals↗
PubMed2004

Chimeras of the homing endonuclease PI-SceI and the homologous Candida tropicalis intein: a study to explore the possibility of exchanging DNA-binding modules to obtain highly specific endonucleases with altered specificity.

Homing endonucleases are extremely specific endodeoxyribonucleases. In vivo, these enzymes confer mobility on their genes by inducing a very specific double-strand cut in cognate alleles that lack the cooling sequence for the homing endonuclease; the cellular repair of the double-strand break with the endonuclease-containing allele as a template leads to integration of the endonuclease gene, completing the homing process. As a result of their extreme sequence specificity, homing endonucleases are promising tools for genome engineering. For this purpose, it is desirable to design enzymes with defined new specificities. To analyse which DNA-binding elements are potential candidates for use in the design of enzymes with modified or even new specificity, we produced several chimeric proteins derived from the Saccharomyces cerevisiae VMA1 intein (PI-SceI) and the related Candida tropicalis VMA1 intein. Although the mature Candida intein is devoid of endonucleolytic activity, the exchange of two DNA-binding modules of PI-SceI with the homologous elements from the Candida intein results in an active endonuclease. The low sequence homology in these modules indicates that different protein-DNA contacts are responsible for the recognition of related DNA sequences. This flexibility in DNA recognition should, in principle, allow endonucleases to be produced with new specificities useful for genome engineering.

Amino Acid Sequence↗
PubMed1994

DNA strand breakage during physiological apoptosis of the embryonic chick lens: free 3' OH end single strand breaks do not accumulate even in the presence of a cation-independent deoxyribonuclease.

Epithelial cells from the lens equator differentiate into elongated fiber cells. In the final steps of differentiation, the chromatin appears quite condensed and chromatin breakdown into nucleosomes occurs. DNA breaks due to an endodeoxyribonuclease activity corresponding to at least two polypeptides of 30 and 40 kDa have been identified. To identify the nature and the developmental appearance of initial breaks, nick translation reaction was followed both biochemically and in situ in fiber and epithelial cells from chick embryonic lenses. There is no accumulation of single-strand breaks (SSB) with 3'OH ends in lens fiber cells during embryonic development. Such damage can be increased in these cells by treatment with DNAase I indicating the absence of an inhibitor of the nick translation reaction in fiber cells. However, there are indications of the presence of DNA breaks with blocked termini when the phosphatase activity of nuclease P1 is used. The presence of breaks is also indicated by the large amounts of (ADP-ribose)n found in lens fibers particularly at 11 days of embryonic development (E11) as ADP-ribosyl transferase binds to and is activated by DNA strand breaks. Incubation of lens cells in vitro, which causes nucleosomal fragmentation only in fiber cells, produces SSB with 3'OH ends in both epithelia and fibers. Incubation for short periods, observed in experiments in situ, induces SSB first in the central fiber nuclei, which are late in differentiation. This may indicate that these SSB play a physiological role. Long incubations produce larger numbers of SSB in epithelia than fibers. The SSB in the fibers may have been converted into double-strand breaks (D SB), seen as nucleosomal fragments, and therefore no longer act as substrates for nick translation. The nuclease activity responsible for SSB production is independent of divalent cations and could be implicated in lens terminal differentiation.

Adenosine Diphosphate Ribose↗
PubMed1987

Mini-Mu transposition of bacterial genes on the transmissible plasmid.

Using the pRM30 plasmid, an Aps deletion derivative of broad host range plasmid RP4 with integrated new miniMu 5 (11 kb), we followed the transfer of Escherichia coli chromosomal genes to the recipient strain. The miniMu 5-mediated transposition of chromosomal genes occurs onto the plasmid with integrated miniMu 5 rather than onto the "recipient" plasmid pNH602. The plasmid DNA in recipient cells was detected by electrophoresis. One of the acquired hybrid plasmids pTB2 was analyzed genetically and by restriction endodeoxyribonuclease digestion. A structure consisting of miniMu-chromosomal segment-miniMu as a product of Mu-mediated transposition was detected.

Chromosome Deletion↗