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PubMed1977

Endonuclease from Escherichia coli that acts specifically upon duplex DNA damaged by ultraviolet light, osmium tetroxide, acid, or x-rays.

An endonuclease which is active upon DNA exposed to ultraviolet light at a photoproduct other than thymine dimers has been extensively purified from Escherichia coli. The small (2.7 S) enzyme is active in the presence of EDTA, has a neutral pH optimum, and is inhibited by tRNA and 1 M NaCl. It has no detectable exonuclease, DNA-N-glycosidase, or ribonuclease activities. The enzyme also nicks duplex DNA exposed to OsO4, x-rays, or acid, but it does not act upon undamaged DNA or irradiated single-stranded DNA. The majority of sites of action in DNA exposed to ultraviolet light or OsO4 appear to be alkali-stable, but those in DNA exposed to x-rays or acid are not. The incisions created by the endonuclease contain 5'-phosphate termini. The enzyme is possibly the same as E. coli endonuclease III described by Radman (Radman, M. (1976) J. Biol. Chem. 251, 1438-1445), but it is distinguishable from the other endodeoxyribonucleases described from that organism.

Bacteriophages↗

PubMed1991

Solid-phase synthesis of oligo(2'-deoxyxylonucleotides) and PCR amplification of base-modified DNA fragments.

1-(2'-Deoxy-beta-D-threo-pentofuranosyl)thymine (xTd) and -adenine (xAd) were converted into their appropriately protected 3'-phosphonates 1a, 2a as well as their 2-cyanoethyl phosphoramidites 1b, 2b. These compounds were used for solid-phase syntheses of the oligo(2'-deoxy-beta-D-xylonucleotides) 5-8. Structural properties and behavior against nucleases is described. Apart from oligo(2'-deoxyxylonucleotides) the PCR-amplification of a pUC18 DNA fragment with Taq polymerase was studied in the presence of the 7-deazapurine derivatives of dGTP, dATP, and dITP. The incorporation efficiency of the modified compounds was compared with those of the parent nucleotides. 7-Deaza-2'-deoxyguanosine protected the DNA-fragment from hydrolysis by the restriction endodeoxyribonuclease Eco RI, Pst I, Bam HI, and Sma I if the nucleoside was located within the recognition site.

Adenosine↗

PubMed1986

An endonuclease from Chlamydomonas reinhardtii that cleaves the sequence TATA.

An endodeoxyribonuclease, designated CreI, was purified 16,000-fold from zygotes of the eukaryote Chlamydomonas reinhardtii. CreI preferentially attacks the sequence TATA producing double strand breaks with 3'-phosphomonoester and 5'-hydroxyl termini. The endonuclease has an Mr = 27,000 and requires Ca2+ at pH 7.5 for optimal activity.

Base Sequence↗

PubMed1979

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.

DNA-Directed DNA Polymerase↗

PubMed1980

Molecular structure of adeno-associated virus variant DNA.

When lysates of human cells, infected jointly with the defective parvovirus, adeno-associated virus (AAV), and a helper adenovirus, are banded to equilibrium in CsCl buoyant density gradients, virus particles of various densities are obtained. Infectious AAV particles mainly band at a density of 1.41 g/cm3 with a minor component at 1.45 g/cm3. Noninfectious AAV particles band at densities between 1.41 and 1.32 g/cm3. We have analyzed, by mapping with site-specific endodeoxyribonucleases, the molecular structure of the variant AAV DNA molecules obtained from these light density particles. The size of variant DNA molecules ranged from 100 to 3% of genome length. In general, the variant DNAs are deleted for internal regions but retain the genome termini. Some of the variant DNAs appear to be cross-linked, spontaneously renaturing molecules having structures analogous to replicating forms of AAV DNA.

Adenoviruses, Human↗

PubMed1980

Purification, subunit structure, and serologicai analysis of calf thymus ribonuclease H I.

Calf thymus ribonuclease H I (for nomenclature, see Büsen, W., and Hausen, P. (1975) Eur. J. Biochem. 52, 179-190) has been purified to near homogeneity. The large scale purification procedure results in a 1,000-fold enrichment of enzyme protein over the crude extract. The enzyme has a molecular weight of about 80,000, and S value of about 5, and an isoelectric point of about 4.9 under nondenaturing conditions. The purified enzyme sample contains two forms of ribonuclease H I, possibly isozymes, named ribonuclease H I 1 and ribonuclease H I 2. They can be activated by Mn2+ or Mg2+ ions. The most highly purified fraction is composed of four polypeptides named A, B, C, and D with molecular weights of 31,6000, 26,6000, 24,800, and 24,300, respectively. Polypeptides A, C, and D are acidic, whereas Polypeptide B is basic. Each form consists of three polypeptides. Ribonuclease H I 1 and ribonuclease H I 2 have Polypeptides A and B in common and differ from each other in the third. The data are consistent with a trimeric (A, B, C/D) or tetrameric (A, B2, C/D) structure for calf thymus ribonuclease H I. When alkalisensitive supercoiled DNA molecules containing ribonucleotides covalently inserted in one of the DNA strands are used as substrate, the products of the reaction are relaxed circles; thus, ribonuclease H I has an endonucleolytic mode of action. The final preparation is free of ribonuclease, and also of endodeoxyribonuclease activity single- and double-stranded DNA. Rabbit antiserum raised against the most highly purified calf thymus ribonuclease H I specifically precipitates the Polypeptides A, B, C, and D and inhibits the Mn2+ - and Mg2+ -dependent enzyme activities to more than 90%. Whereas a typical monophasic neutralization curve is obtained with Mn2+ activation, the neutralization curve observed with Mg2+ is biphasic. These results and several other differences between the Mn2+ - and the Mg2+ -dependent activities of the ribonucleases H I seem best explained by a hypothesis in which the enzymes exist in two different conformations depending on the type of divalent cation activation. The antiserum neutralizes ribonuclease H I but not the other known calf thymus ribonuclease H activities (IIa; IIb), demonstrating that the different ribonuclease H activities in calf thymus are serologically distinct. Ribonuclease H I is localized in the cell nucleus as visualized by immunofluorescent staining of bovine cells.

Animals↗

PubMed1981

Purification and characterization of an apurinic/apyrimidinic endonuclease from HeLa cells.

An endodeoxyribonuclease from HeLa cells acting on apurinic/apyrimidinic (AP) sites has been purified to apparent homogeneity as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The presence of Triton X-100 was necessary throughout the purification for stabilization and stimulation of activity. The endonuclease has an apparent native molecular weight of 32,000 determined by molecular sieving and an apparent subunit molecular weight of 41,000 as judged by its electrophoretic mobility in SDS-polyacrylamide gels. The activity has an absolute requirement for Mg2+ or Mn2+ and a broad pH optimum between 6.7 and 9.0 with maximal activity near pH 7.5. The enzyme has no detectable exonuclease activity, nor any endonuclease activity on untreated duplex or single-stranded DNA. It is inhibited by adenine, hypoxanthine, adenosine, AMP, ADP-ribose, and NAD+, but it is unaffected by caffeine, the pyrimidine bases, ADP, ATP, or NADH. The use of a variety of damaged DNA substrates provided no indication that the enzyme acts on other than AP sites. The enzyme appears to cleave AP DNA so as to leave deoxyribose-5-phosphate at the 5' terminus and a 3'-OH at the 3' terminus; it also removes deoxyribose-5-phosphate from AP DNA which has deoxyribose at the 3' terminus. Specific antibody has been produced in rabbits which interacts only with a 41,000-dalton protein present in the purified enzyme (presumably the enzyme itself), as well as with partially purified AP endonuclease fractions from human placenta and fibroblasts.

Animals↗

PubMed1983

Excision repair and DNA synthesis with a combination of HeLa DNA polymerase beta and DNase V.

The ability of HeLa DNA polymerases to carry out DNA synthesis from incisions made by various endodeoxyribonucleases which recognize or form baseless sites in DNA was examined. DNA polymerase beta carried out limited strand displacement synthesis from 3'-hydroxyl nucleotide termini made by HeLa apurinic/apyrimidinic (AP) endonuclease II at the 5'-side of apurinic sites. Escherichia coli endonuclease III incises at the 3'-side of apurinic sites to produce nicks with 3'-deoxyribose termini which did not efficiently support DNA synthesis with beta-polymerase. However, these nicks could be activated to support limited DNA synthesis by HeLa AP endonuclease II, an enzyme which removes the baseless sugar phosphate from the 3'-termini, thus creating a one-nucleotide gap. With dGTP as the only nucleoside triphosphate present, the beta-polymerase catalyzed one-nucleotide DNA repair synthesis from those gaps which lacked dGMP. In contrast, HeLa DNA polymerase alpha was unreactive with all of the above incised DNA substrates. Larger patches of DNA synthesis were produced by nick translation from one-nucleotide gaps with HeLa DNA polymerase beta and HeLa DNase V. Moreover, incisions made by E. coli endonuclease III were activated to support DNA synthesis by the DNase V which removed the 3'-deoxyribose termini. HeLa DNase V also stimulated both the rate and extent of DNA synthesis by DNA polymerase beta from AP endonuclease II incisions. In this case the baseless sugar phosphate was removed from the 5'-termini, and nick translational synthesis occurred. Complete DNA excision repair of pyrimidine dimers was achieved with the beta-polymerase, DNase V, and DNA ligase from incisions made in UV-irradiated DNA by T4 UV endonuclease and HeLa AP endonuclease II. Such incisions produce a one-nucleotide gap containing 3'-hydroxyl nucleotide and 5'-thymine: thymidylate cyclobutane dimer termini. DNase V removes pyrimidine dimers primarily as a dinucleotide and then promotes nick translational DNA synthesis.

DNA Ligases↗

PubMed1993

A broad-specific alkaline DNase from rat brain with a putative role in DNA excision repair.

An alkaline endodeoxyribonuclease from rat brain has been purified to near homogeneity. The purified enzyme showed a M.Wt. of 54 Kd on SDS-PAGE and does not require metal ion for activity and thus differs from classical DNase I. No preference towards any particular form of calf thymus DNA (native, denatured, undamaged and damaged by exposure to UV, H2O2 and OsO4 and depurination) was noticed. However, with supercoiled pBR 322 plasmid DNA as substrate, higher activity was exhibited towards UV irradiated and depurinated forms. It is suggested that this DNase may be a 'housekeeping' enzyme to detect any conformational distortion in DNA and initiate excision repair.

Animals↗

PubMedDate not supplied

[Induction of synthesis of extracellular DNAase from Proteus mirabilis under the effect of compounds, blocking DNA replication].

The effects of mitomycin C and nalidixic acid on the biosynthesis of extracellular endodeoxyribonuclease in Proteus mirabilis have been studied. The presence of both antibiotics in the periodic and short-time cultures of washed off cells has increased both the activity of the DNAse and protein yield in cultural liquid and bacterial cells. PAGE-electrophoresis has shown the effect of mitomycin C to increase or induce the synthesis a large number of Proteus mirabilis extracellular proteins.

DNA Replication↗

PubMed1979

Endonuclease activity of purified RNA-directed DNA polymerase from avian myeloblastosis virus.

Highly purified preparations of RNA-directed DNA polymerase from avian myeloblastosis virus (AMV) contain a Mn2+-activated endonuclease activity capable of nicking supercoiled DNA. This endonuclease activity co-sediments in glycerol gradients with the alphabeta form of AMV DNA polymerase, and co-chromatographs with DNA polymerase activity on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. It is also present in AMV alphabeta-DNA polymerase purified by electrophoresis through nondenaturing polyacrylamide gels and subsequently chromatographed on poly(C)-agarose. alphabeta-associated endonuclease is co-immunoprecipitated with DNA polymerase activity by antiserum directed against alphabeta holoenzyme. The alpha form of AMV DNA polymerase lacks this activity. In its enzymatic properties, alphabeta-associated endonuclease resembles the endodeoxyribonuclease activity associated with the AMV p32 protein, which has been shown to be structurally related to the beta (but not the alpha) subunit of AMV DNA polymerase.

Avian Leukosis Virus↗

PubMed2003

DNA fragmentation decreased in schizophrenia but not bipolar disorder.

BACKGROUND: Apoptosis is thought to play a role in neuronal pathology in schizophrenia and bipolar disorder. METHODS: To test this hypothesis, the Klenow method for in situ end-labeling of single-stranded DNA breaks was applied to anterior cingulate cortex from 18 healthy controls, 18 schizophrenic subjects, and 10 bipolar subjects. RESULTS: An unexpected reduction (71%) in Klenow-positive nuclei was found in schizophrenic but not in bipolar cortexes. CONCLUSIONS: To our knowledge to date, this is the first demonstration that there is much less DNA fragmentation in individuals with schizophrenia than in healthy controls and bipolar subjects, which raises a key question as to whether this alteration represents an adaptive or nonadaptive change in the regulation of intracellular signaling and mitochondrial oxidative pathways associated with apoptosis.

Adolescent↗

PubMed2026

Prevalence of the Predisposing Gene MBD4 for Uveal Melanoma.

IMPORTANCE: MBD4 monoallelic germline pathogenic and likely pathogenic variants have recently been identified as predisposing to uveal melanoma, a rare primary intraocular tumor, with an estimated 9.15-fold increased risk of developing the disease for pathogenic variant carriers. OBJECTIVE: To assess the risk of developing uveal melanoma for carriers of the MBD4 monoallelic germline pathogenic variant. DESIGN, SETTING, AND PARTICIPANTS: In a case series involving 896 individuals, including 319 who were previously evaluated, germline target-sequencing of MBD4 was offered to every new patient with uveal melanoma at Curie Institute from February 2021 to September 2025. Non-Finnish European participants from the Genome Aggregation Database were used as a reference population. EXPOSURE: Diagnosis of uveal melanoma genetic predisposition. MAIN OUTCOMES AND MEASURES: Prevalence of MBD4 variants. RESULTS: A total of 23 of 896 patients were identified as carrying an MBD4 germline pathogenic or likely pathogenic variant, corresponding to a relative risk of 31.44 (95% CI, 18.18-53.00) of developing uveal melanoma compared with the general population (2-sided Fisher exact test, P < .001). CONCLUSIONS AND RELEVANCE: These findings confirm that MBD4 is an important predisposing gene to uveal melanoma in the French population. This reinforces a strategy of broad patient screening given the therapeutic implications and the consequences of genetic counseling.

Humans↗

PubMed1997

Analysis of an 11.6 kb region from the right arm of chromosome VII of Saccharomyces cerevisiae between the RAD2 and the MES1 genes reveals the presence of three new genes.

Sequence analysis of an 11,628 bp DNA segment from the right arm of Saccharomyces cerevisiae chromosome VII revealed the presence of the 5' end of the RAD2 gene, the MES1 gene and six open reading frames (ORFs) each longer than 300 bp. Four of these ORFs are expressed genes, as indicated by transcript analysis. One of them, YGR261c, which specifies a putative beta-adaptine, corresponds to gene YKS5, which has recently been identified as a suppressor of loss of casein kinase 1 function. The remaining three ORFs are new genes; of these, YGR260w encodes a protein showing similarity to the S. cerevisiae allantoate permease and YGR262c specifies a putative protein kinase.

Blotting, Northern↗

PubMed1997

Analysis of the structure of a natural alternating d(TA)n sequence in yeast chromatin.

We address here the question of the in vivo structure of a natural alternating d(TA)n sequence found at the 3' region of the Saccharomyces cerevisiae FBP1 gene. This sequence consists of 13 TA pairs interrupted by a TT dinucleotide in the middle of the tract. Previous experiments with cruciform-specific nucleases S1 and Endonuclease VII demonstrated the presence in vitro of a cruciform in this region. We also showed this region to be part of a nuclease hypersensitive site flanked by nucleosomes in yeast chromatin. Here we demonstrate, by means of S1 in vivo footprinting, that in yeast plasmids also adopts in vivo a non B-DNA structure where is not a cruciform. A theoretical analysis of this region that it contains a site susceptible to superhelical stress duplex destabilization. The locations and conditions under which alternative structures form in the wild-type sequence and in deletion mutants agree with these theoretical predictions, suggesting that some kind of denaturation is the alternative structure adopted by the sequence in vivo. This suggests that negative superhelical stress sufficient for local denaturation exists in nucleosomal DNA. We also demonstrate by micrococcal nuclease digestions that the deletion of the alternating d(TA)n sequence modifies the chromatin hypersensitive site but does not affect nucleosome positioning.

Base Sequence↗

PubMed1997

DNA recognition by structure-selective nucleases.

The nucleases discussed in this review show little sequence specificity but instead recognize certain structural features of their respective DNA substrates. The level of their structural selectivity ranges from simple discrimination between single- and double-stranded DNA (nucleases P1 and S1), the recognition of helical parameters like groove width and flexibility (DNase I), the recognition of helical distortions caused by abasic sites (exonuclease III, HAP1), to the recognition of specialized structures like flap DNA (5'-nucleases of eukaryotes, phages, and eubacterial DNA polymerases) and four-way junctions (T4 endonuclease VII, RuvC). The discussion is focused on the structural basis of the recognition process. In most cases the available x-ray structures of the nucleases and/or their DNA complexes have revealed the presence of structural motifs explaining the observed structural selectivity.

Binding Sites↗

PubMed1996

Pathophysiology of oligodendroglial excitotoxicity.

Oligodendrocyte-like cells (OLD) derived from the rat oligodendroglial precursor line, CG-4, express Ca(2+)-permeable non-methyl-D-aspartate glutamate receptor channels (GluR). Exposure to kainate, an L-glutamate analogue, markedly elevates OLC Ca2+ influx and cytosolic [Ca2+], and results in damage to both OLC plasma membrane and OLC nuclear DNA. Two observations indicate that kainate-induced OLC internucleosomal DNA nicking is not simply a delayed consequence of cell necrosis: 1) there is no temporal lag between onset of plasma membrane injury and of DNA nicking; and 2) aurintricarboxylic acid, an endonuclease inhibitor, blocks kainate-induced damage to the plasma membrane. N-acetyl-L-cysteine also inhibits OLC kainate injury, suggesting that reactive oxygen species participate in OLC excitotoxicity. Kainate-induced OLC Ca2+ influx and excitotoxicity are blocked by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), indicating that these kainate effects are mediated by AMPA-GluR. AMPA and L-glutamate fail to elicit OLC damage unless cyclothiazide, an AMPA-GluR desensitization blocker, is present. OLC express both the "flip" and "flop" forms of GluR2, GluR3, and GluR4 mRNAs, but neither flip nor flop GluR1 mRNA. These data, together with the restriction of the desensitization-blocking activity of cyclothiazide to GluR containing flip-encoded GluR subunits, and the sharply diminished Ca2+ permeability of GluR containing edited GluR2, suggest OLC excitotoxicity is mediated by AMPA-GluR that contain flip GluR3 and/or flip GluR4 protein subunits, but neither flip nor flop GluR2 protein subunits. Rapid desensitization of these GluR is likely to be important in protecting cells of the oligodendroglial lineage from excitotoxicity.

Acetylcysteine↗

PubMed1997

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase II alpha associated with nuclear matrices from wild-type and drug-resistant chinese hamster ovary cell lines.

Topo II alpha is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo II alpha associated with the nuclear matrix in drug-resistant SMR16 and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo II alpha in individual nuclear matrices. There are significant variations in topo II alpha amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix-associated topo II alpha than the resistant cell line matrices. Nuclear matrix-associated topo II alpha from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo II alpha in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures.

Animals↗