Purification and properties of yeast redoxyendonuclease.
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Base Sequence↗
Biomedical subjects
P W Doetsch
Publications and source records attributed to P W Doetsch.
Effects of abasic sites and DNA single-strand breaks on prokaryotic RNA polymerases.
Abasic sites are thought to be the most frequently occurring cellular DNA damage and are generated spontaneously or as the result of chemical or radiation damage to DNA. In contrast to the wealth of information that exists on the effects of abasic sites on DNA polymerases, very little is known about how these lesions interact with RNA polymerases. An in vitro transcription system was used to determine the effects of abasic sites and single-strand breaks on transcriptional elongation. DNA templates were constructed containing single abasic sites or nicks placed at unique locations downstream from two different promoters and were transcribed by SP6 and Escherichia coli RNA polymerases. SP6 RNA polymerase is initially stalled at abasic sites with subsequent, efficient bypass of these lesions. E. coli RNA polymerase also bypassed abasic sites. In contrast, single-strand breaks introduced at abasic sites completely blocked the progression of both RNA polymerases. Sequence analysis of full-length transcripts revealed that SP6 and E. coli RNA polymerases insert primarily, if not exclusively, adenine residues opposite to abasic sites. This finding suggests that abasic sites may be highly mutagenic in vivo at the level of transcription.
Base Composition↗
A eukaryotic DNA glycosylase/lyase recognizing ultraviolet light-induced pyrimidine dimers.
Cyclobutane pyrimidine dimers (CPDs) are the predominant product of photodamage in DNA after exposure of cells to ultraviolet light and are cytotoxic, mutagenic and carcinogenic in a variety of cellular and animal systems. In prokaryotes, enzymes and protein complexes have been characterized that remove or reverse CPDs in DNA. Micrococcus luteus and T4 phage-infected Escherichia coli contain a specific N-glycosylase/apurinic-apyrimidinic lyase that catalyses a two-step DNA incision process at sites of CPDs, thus initiating base excision repair of these lesions. It is well established that CPDs are recognized and removed from eukaryotic DNA by excision repair processes but very little information exists concerning the nature of the proteins involved in CPD recognition and DNA incision events. We report here that an enzyme functionally similar to the prokaryotic N-glycosylase/apurinic-apyrimidinic lyases exists in Saccharomyces cerevisiae. To our knowledge, this is the first time such an activity has been found in a eukaryote and is also the first example of an organism having both direct reversal and base excision repair pathways for the removal of CPDs from DNA.
Base Sequence↗
Purification and properties of nuclease SP.
Single-strand-specific nucleases are a diverse and important group of enzymes that are able to cleave a variety of DNA structures present in duplex molecules. Nuclease SP, an enzyme from spinach, has been purified to apparent homogeneity, allowing for the unambiguous characterization of a number of its physical properties as well as its DNA strand cleavage specificities. The effects of ionic strength, pH, divalent metal cations, and temperature on nuclease SP activity have been examined in detail. Nuclease SP was found to be quite thermostable and could be stimulated by Co2+. In addition, the cleavage of UV-damaged and undamaged supercoiled plasmid substrates under a variety of conditions suggests that at least two types of structures are recognized and processed by nuclease SP: UV photoproduct-induced distortions and unwound "nuclease hypersensitive sites". These studies indicate that nuclease SP is functionally related to other single-strand-specific nucleases and is a potential enzymatic tool for probing and manipulating various types of DNA structures.
Electrophoresis, Polyacrylamide Gel↗
DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs.
A number of eukaryotic DNA binding proteins have been isolated by screening phage expression libraries with DNA probes containing the binding site of the DNA-binding protein. This methodology was employed here to isolate clones of the factor that interacts with the W box element of the human major histocompatibility complex HLA-DQB gene. Surprisingly, several cDNA clones of YB-1, a cDNA clone that was previously isolated with a CCAAT element-containing sequence were found. Independently, the screening of phage expression libraries with depurinated DNA resulted in the isolation of YB-1 and dbpA, a previously isolated cDNA that has homology to YB-1. Additional characterization of YB-1 showed that it bound a wide variety of DNA sequences and suggested that the binding of this protein is promiscuous. Furthermore, we show that both YB-1 and dbpA bind to depurinated DNA better than undamaged DNA and that the extent of specificity of binding is influenced by Mg2+. Due to the lack of sequence specificity and high degree of binding to depurinated DNA, we suggest that these proteins might be involved in chromosome functions such as maintenance of chromatin structure or DNA repair that do not require sequence-specific binding.
Base Sequence↗
Bleomycin A2 and B2 purification by flash chromatography for chemical and biochemical studies.
The clinically used formulation of the anticancer antibiotic, Blenoxane, is a mixture of bleomycin congeners. A new approach to separating the major A2 and B2 congeners has been developed utilizing the flash chromatography technique. A 5-6-inch column of fine mesh silica gel with a solvent system of 1% ammonium formate:methanol (2:3) was used. Low air pressure was applied to the column to increase the flow rate such that separation was complete in approximately 20 min. Reverse phase size exclusion gravity chromatography with Sephadex G-15 column bedding was an effective, rapid procedure for removal of the 1% ammonium formate, the lowest percentage practical for separating the bleomycins. This separation approach does not damage the antibiotics, as demonstrated by NMR spectroscopy, thin layer chromatography, and DNA cleaving activity. Although not as useful for detection of trace amounts of the drug in biological systems as some of the known HPLC methods, this method is excellent for separating large quantities of the drug (8-32 mg) in order to obtain congeners pure enough for synthetic, biochemical, and biophysical studies.
Bleomycin↗
Damage specific mammalian endonucleases.
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Animals↗
Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites.
Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of beta-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins.
Animals↗
Partial purification and characterization of an endonuclease from spinach that cleaves ultraviolet light-damaged duplex DNA.
An endonuclease that cleaves ultraviolet light (UV)-damaged, supercoiled plasmid DNA was partially purified from spinach leaves (Spinacia oleracea) by a series of column chromatography steps. Dialysis of the enzyme against EDTA resulted in a greater than 90% loss of activity which could be fully restored following the addition of Zn2+, suggesting that divalent cations are associated with the active enzyme. The spinach endonuclease cleaved duplex, UV-damaged, end-labelled DNA of defined sequence at positions of adenine in the presence of salt (KH2PO4 or NaCl) concentrations of 50 mM or higher. Cleavage of UV-irradiated DNA was dose-dependent and increased steadily within a fluence range of 50-10,000 J/m2. The UV damage requirement and adenine cleavage specificity could be eliminated with lower salt concentrations (0-25 mM), suggesting that the endonuclease recognizes and incises single-stranded DNA. The properties of this enzyme, which we have termed nuclease SP, suggest that it may mediate a role in DNA repair and/or recombination processes in spinach.
DNA↗
Nuclease SP: a novel enzyme from spinach that incises damaged duplex DNA preferentially at sites of adenine.
A novel endonuclease has been isolated from extracts of spinach leaves (Spinacia oleracea). The enzyme has been purified by a series of column chromatography steps and has a molecular size of approximately 43,000 daltons. The spinach endonuclease cleaved double stranded DNA damaged by ultraviolet light or cis-diamminedichloroplatinum (II) primarily at sites of adenine when end-labelled DNA fragments of defined sequence were employed as substrates. The nature of the structural distortion contained in damaged, duplex DNA appears to be an important determinant for endonuclease cleavage. DNA helical distortions produced by UV light-induced (6-4) pyrimidine-pyrimidone photoproducts, but not cyclobutane pyrimidine dimers are recognized by the enzyme. The DNA cleavage products generated by the enzyme contain 3'-hydroxyl and 5'-phosphoryl termini. Single stranded DNA and RNA are hydrolyzed by the spinach endonuclease. This enzyme, which we call nuclease SP, is similar in several respects to other single-strand-specific nucleases such as N. crassa and mung bean nucleases and may function in DNA repair and/or recombination events in spinach cells. Nuclease SP should be a useful tool for the analysis of (6-4) photoproducts occurring in duplex DNA.
Base Sequence↗
Yeast redoxyendonuclease, a DNA repair enzyme similar to Escherichia coli endonuclease III.
A DNA repair endonuclease (redoxyendonuclease) was isolated from bakers' yeast (Saccharomyces cerevisiae). The enzyme has been purified by a series of column chromatography steps and cleaves OsO4-damaged, double-stranded DNA at sites of thymine glycol and heavily UV-irradiated DNA at sites of cytosine, thymine, and guanine photoproducts. The base specificity and mechanism of phosphodiester bond cleavage for the yeast redoxyendonuclease appear to be identical with those of Escherichia coli endonuclease III when thymine glycol containing, end-labeled DNA fragments of defined sequence are employed as substrates. Yeast redoxyendonuclease has an apparent molecular size of 38,000-42,000 daltons and is active in the absence of divalent metal cations. The identification of such an enzyme in yeast may be of value in the elucidation of the biochemical basis for radiation sensitivity in certain yeast mutants.
Base Sequence↗
Wavelength dependence for human redoxy-endonuclease-mediated DNA cleavage at sites of UV-induced photoproducts.
Redoxy-endonuclease, an enzyme present in human and other cells, recognizes monobasic photoproducts that occur primarily at sites of cytosine following UV-irradiation of DNA at 254 nm. The wavelength dependence for formation of these photoproducts was determined using end-labeled DNA fragments of defined sequence irradiated with monochromatic light ranging from 254-360 nm as substrates for redoxy-endonuclease partially purified from HeLa cells. The base specificity and extent of DNA cleavage were determined by analysis of the enzyme-generated DNA scission products on DNA sequencing gels. Maximal incision at sites of cytosine and thymine was observed at 280 nm, suggesting that these photoproducts may be relevant with respect to the biological effects of solar radiation.
DNA↗
Thymine glycol-DNA glycosylase/AP endonuclease of CEM-C1 lymphoblasts: a human analog of Escherichia coli endonuclease III.
A thymine glycol-DNA glycosylase/AP endonuclease has been identified in human CEM-C1 lymphoblasts. The enzyme is active in the absence of divalent cations and has an apparent molecular size of approximately 60,000 daltons. The enzyme releases thymine glycol from osmium tetroxide-damaged DNA via an N-glycosylase activity and is associated with an endonuclease activity that mediates phosphodiester bond cleavage at sites of thymine glycol and apurinic sites. We propose that this enzyme, which we call redoxyendonuclease, is the human analog of a bacterial enzyme, E. coli endonuclease III, that recognizes oxidative DNA damage.
Cations, Divalent↗
A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines.
We have compared the sites of nucleotide incision on DNA damaged by oxidizing agents when cleavage is mediated by either Escherichia coli endonuclease III or an endonuclease present in bovine and human cells. E. coli endonuclease III, the bovine endonuclease isolated from calf thymus, and the human endonuclease partially purified from HeLa and CEM-C1 lymphoblastoid cells incised DNA damaged with osmium tetroxide, ionizing radiation, or high doses of UV light at sites of pyrimidines. For each damaging agent studied, regardless of whether the E. coli, bovine, or human endonuclease was used, the same sequence specificity of cleavage was observed. We detected this endonuclease activity in a variety of human fibroblasts derived from normal individuals as well as individuals with the DNA repair deficiency diseases ataxia telangiectasia and xeroderma pigmentosum. The highly conserved nature of such a DNA damage-specific endonuclease suggests that a common pathway exists in bacteria, humans, and other mammals for the reversal of certain types of oxidative DNA damage.
Animals↗
Mechanism of action of a mammalian DNA repair endonuclease.
The mechanism of action of a DNA repair endonuclease isolated from calf thymus was determined. The calf thymus endonuclease possesses a substrate specificity nearly identical with that of Escherichia coli endonuclease III following DNA damage by high doses of UV light, osmium tetroxide, and other oxidizing agents. The calf thymus enzyme incises damaged DNA at sites of pyrimidines. A cytosine photoproduct was found to be the primary monobasic UV adduct. The calf thymus endonuclease and E. coli endonuclease III were found to possess similar, but not identical, DNA incision mechanisms. The mechanism of action of the calf thymus endonuclease was deduced by analysis of the 3' and 5' termini of the enzyme-generated DNA scission products with DNA sequencing methodologies and HPLC analysis of the material released by the enzyme following DNA damage. The calf thymus endonuclease removes UV light and osmium tetroxide damaged bases via an N-glycosylase activity followed by a 3' apurinic/apyrimidinic (AP) endonuclease activity. The calf thymus endonuclease also possesses a novel 5' AP endonuclease activity not possessed by endonuclease III. The product of this three-step mechanism is a nucleoside-free site flanked by 3'-and 5'-terminal phosphate groups. These results indicate the conservation of both substrate specificity and mechanism of action in the enzymatic removal of oxidative base damage between prokaryotes and eukaryotes. We propose the name redoxy endonucleases for this group of enzymes.
Animals↗
Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage.
The substrate specificity of a calf thymus endonuclease on DNA damaged by UV ligh, ionizing radiation, and oxidizing agents was investigated. End-labeled DNA fragments of defined sequence were used as substrates, and the enzyme-generated scission products were analyzed by using DNA sequencing methodologies. The enzyme was shown to incise damaged DNA at pyrimidine sites. The enzyme incised DNA damaged with UV light, ionizing radiation, osmium tetroxide, potassium permanganate, and hydrogen peroxide at cytosine and thymine sites. The substrate specificity of the calf thymus endonuclease was compared to that of Escherichia coli endonuclease III. Similar pyrimidine base damage specificities were found for both enzymes. These results define a highly conserved class of enzymes present in both procaryotes and eucaryotes that may mediate an important role in the repair of oxidative DNA damage.
Animals↗
Cyclobutane pyrimidine dimers and (6-4) photoproducts block polymerization by DNA polymerase I.
Bipyrimidine cyclobutane dimers and 6-4'-(pyrimidin-2'-one)-pyrimidine photoproducts are the major adducts formed in DNA following exposure to ultraviolet light. The relationship between the type and frequency of UV-induced DNA damage and the effects of such damage on DNA replication were investigated. UV-irradiated M13 phage DNA was employed in polymerization reactions with the Kenow fragment of Escherichia coli DNA polymerase I. The locations and frequencies of polymerase termination events occurring within a defined sequence of M13 DNA were compared with measurements of the locations and frequencies of UV-induced DNA damage of the same DNA sequence by using UV-specific enzymatic and chemical methods. The results indicate that both cyclobutane dimers and (6-4) photoproducts quantitatively block polymerization by DNA polymerase I.
Coliphages↗
Structural determination of the ultraviolet light-induced thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct.
Ultraviolet light induces damage to DNA, with the majority of the damage expressed as the formation of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts. The (6-4) photoproducts have been implicated as important UV light-induced premutagenic DNA lesions. The most abundant of the (6-4) products is the thymine-cytosine pyrimidine-pyrimidone (6-4) photoproduct, or TC (6-4) product. The structure of the TC (6-4) product was deduced by proton NMR, IR, and fast atom bombardment mass spectroscopy, and the product was found to differ from the previously described photoadduct, Thy(6-4)Pyo, by the presence of an amino group at the 5 position of the 5' pyrimidine. The implications of this structure on DNA base pairing and the induction of ultraviolet light-induced mutations are discussed.
Cytosine↗