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R P Cunningham

Publications and source records attributed to R P Cunningham.

At least 55 records · Page 3Linked to original sources
PubMed1992

Transfection of the Escherichia coli nth gene into radiosensitive Chinese hamster cells: effects on sensitivity to radiation, hydrogen peroxide, and bleomycin sulfate.

The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.

Animals↗
PubMed1991

Stereochemical studies of the beta-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys.

The DNA strand cleavage reaction catalyzed by endonuclease III from Escherichia coli (endo III) on the 3'-side of aldehyde abasic sites proceeds by a syn beta-elimination involving abstraction of the 2'-pro-S proton and formation of a trans alpha,beta-unsaturated aldose product; we previously reported the same stereochemical course for the reaction catalyzed by UV endonuclease V from bacteriophage T4 (UV endo V) [Mazumder, A., Gerlt, J. A., Rabow, L., Absalon, M. J., Stubbe, J., & Bolton, P. H. (1989) J. Am. Chem. Soc. 111, 8029-8030]. Since the UV endo V does not contain an 4Fe-4S center, the 4Fe-4S center present in endo III need not be assigned a unique role in the beta-elimination reaction. The beta-elimination reactions that occur under alkaline conditions (0.1 N NaOH) and in the presence of the tripeptide Lys-Trp-Lys proceed by anti beta-elimination mechanisms involving abstraction of the 2'-pro-R proton and formation of a trans alpha,beta-unsaturated aldose product. The different stereochemical outcomes of the enzymatic and nonenzymatic beta-elimination reactions support the hypothesis that the enzyme-catalyzed reactions may involve general-base-catalyzed abstraction of the 2'-pro-S proton by the internucleotidic phosphodiester leaving group.

Aldehydes↗
PubMed1990

Formation and stability of repairable pyrimidine photohydrates in DNA.

Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases [Boorstein et al. (1989) Biochemistry 28, 6164-6170]. Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation. Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C. Uracil hydrate and uracil were also formed in irradiated poly(dG-dC). These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase.

Animals↗
PubMed1990

Enzymatic recognition of DNA modifications induced by singlet oxygen and photosensitizers.

DNA modifications induced either by photosensitization (illumination in the presence of methylene blue) or by chemically generated singlet oxygen (thermal decomposition of an 1,4-etheno-2,3-benzodioxin) are recognized and incised by repair endonucleases present in crude bacterial cell extracts. Only a small fraction of the incised modifications are sites of base loss (AP-sites) sensitive to exonuclease III, endonuclease IV from E. coli or to the UV-endonuclease from M. luteus. Cell extracts from E. coli strains overproducing or defective in endonuclease III recognize the modifications induced by illumination in the presence of methylene blue just as well as do those from wild-type E. coli strains. This indicates that dihydropyrimidine derivatives, which are characteristic of hydroxyl radical-induced DNA modifications, are absent. In contrast, most of the modifications induced are not recognized by a cell extract from a fpg strain defective in formamidopyrimidine-DNA glycosylase FPG protein). Furthermore, incision by a cell extract from an E. coli strain overproducing FPG protein takes place at much lower protein concentration than with the wild-type strain. Experiments with purified FPG protein confirm that this enzyme is responsible for the recognition of singlet oxygen-induced DNA base modifications.

Coliphages↗
PubMed1990

Resolution of Holliday junction analogs by T4 endonuclease VII can be directed by substrate structure.

Endonuclease VII is an enzyme from bacteriophage T4 capable of resolving four-arm Holliday junction intermediates in recombination. Since natural Holliday junctions have homologous (2-fold) sequence symmetry, they can branch migrate, creating a population of substrates that have the branch point at different sites. We have explored the substrate requirements of endonuclease VII by using immobile analogs of Holliday junctions that lack this homology, thereby situating the branch point at a fixed site in the molecule. We have found that immobile junctions whose double-helical arms contain fewer than nine nucleotide pairs do not serve as substrates for resolution by endonuclease VII. Scission of substrates with 2-fold symmetrically elongated arms produces resolution products that are a function of the particular arms that are lengthened. We have confirmed that the scission products are those of resolution, rather than nicking of individual strands, by using shamrock junction molecules formed from a single oligonucleotide strand. A combination of end-labeled and internally labeled shamrock molecules has been used to demonstrate that all of the scission is due to coordinated cleavage of DNA on opposite sides of the junction, 3' to the branch point. Endonuclease VII is known to cleave the crossover strands of Holliday junctions in this fashion. The relationship of the long arms to the cleavage direction suggests that the portion of the enzyme which requires the minimum arm length interacts with the pair of arms containing the 3' portion of the crossover strands on the bound surface of the antiparallel junction.

Base Sequence↗
PubMed1990

Construction and analysis of parallel and antiparallel Holliday junctions.

The Holliday junction is a four-stranded DNA intermediate that arises during recombination reactions. We have designed and constructed a set of Holliday junction analogs that model each of the ideal conformations available to a 2-fold symmetric four-arm junction. The strategy used is to connect two arms of a junction molecule with a short tether of thymidines. These DNA molecules share a common core sequence but have different arms that are connected so that each molecule is constrained in either an antiparallel or a parallel structure. For tethered antiparallel molecules the identity of the crossover strands is determined by which arms are connected. Different arm connections gave molecules representing each of the two antiparallel crossover isomers. Two parallel molecules that differ in the length and position of the tether exhibit opposite biases in their choice of crossover strands. Thus, a physical constraint applied at a distance from the branch point can determine the conformation of a junction.

Base Sequence↗
PubMed1989

UV-induced pyrimidine hydrates in DNA are repaired by bacterial and mammalian DNA glycosylase activities.

Escherichia coli endonuclease III and mammalian repair enzymes cleave UV-irradiated DNA at AP sites formed by the removal of cytosine photoproducts by the DNA glycosylase activity of these enzymes. Poly(dG-[3H]dC) was UV irradiated and incubated with purified endonuclease III. 3H-Containing material was released in a fashion consistent with Michaelis-Menten kinetics. This 3H material was determined to be cytosine by chromatography in two independent systems and microderivatization. 3H-Containing material was not released from nonirradiated copolymer. When poly(dA-[3H]dU) was UV irradiated, endonuclease III released 3H-containing material that coeluted with uracil hydrate (6-hydroxy-5,6-dihydrouracil). Similar results are obtained by using extracts of HeLa cells. There results indicate that the modified cytosine residue recognized by endonuclease III and the mammalian enzyme is cytosine hydrate (6-hydroxy-5,6-dihydrocytosine). Once released from DNA through DNA-glycosylase action, the compound eliminates water, reverting to cytosine. This is consistent with the known instability of cytosine hydrate. The repairability of cytosine hydrate in DNA suggests that it is stable in DNA and potentially genotoxic.

Animals↗
PubMed1989

Purification and characterization of Escherichia coli endonuclease III from the cloned nth gene.

The gene which codes for endonuclease III of Escherichia coli has been sequenced. The nth gene was previously subcloned and defined as the gene which led to overproduction of endonuclease III when present on a multicopy plasmid and which created a deficiency in endonuclease III activity when mutated. The nth gene was sequenced and translated into a predicted polypeptide. The molecular weight (23,546), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are excellent agreement with those same properties determined for the purified protein. Thus, the nth gene is the structural gene for endonuclease III. Inspection of the nucleotide sequence reveals that there is an open reading frame immediately upstream of the nth gene, suggesting that it might be part of an operon. There is a region of dyad symmetry which could form a hairpin stem and loop structure if transcribed into RNA characteristic of a rho-dependent terminator downstream from the nth gene. The nth gene of Escherichia coli has been cloned onto a lambda PL expression vector which yields approximately 300-fold overproduction of endonuclease III. We have purified the enzyme to apparent homogeneity using two chromatographic steps. Our purification scheme allowed the preparation of 117 mg of protein from 190 g of E. coli with a 70% yield. The purified protein has both AP endonuclease activity and DNA N-glycosylase activity. The protein has a Stokes radius of 2.25 nm, a sedimentation coefficient of 2.65 S, a molecular weight of 26,300 in the native state and 27,300 in the denatured state, and a frictional ratio of 1.13.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Endonuclease III is an iron-sulfur protein.

Elemental analyses, Mössbauer, and EPR data are reported to show that endonuclease III of Escherichia coli is an iron-sulfur protein. Mössbauer spectra of protein freshly prepared from E. coli grown on 57Fe-enriched medium demonstrate that the native enzyme contains a single 4Fe-4S cluster in the 2+ oxidation state, with a net spin of zero. Upon treatment with ferricyanide, a fraction (less than 25%) of the clusters is oxidized into a state which yields an EPR spectrum near g = 2.01 typical of a 3Fe-4S cluster. The magnetic field dependence of the linear electric field effect verifies this assignment. Electron spin echo modulation on the g = 2.01 form of the protein in deuterated solvent indicates the presence of exchangeable protons in the vicinity of the 3Fe-4S cluster. The data obtained show that the [4Fe-4S]2+ cluster of the native enzyme is resistant to either oxidation or reduction, although photoreduction elicited a g = 1.94 type EPR signal characteristic of a [4Fe-4S]1+ cluster. These studies show that endonuclease III is unique in being both a DNA repair enzyme and an iron-sulfur protein. The function of the 4Fe-4S cluster remains to be established.

Deoxyribonuclease (Pyrimidine Dimer)↗
PubMed1989

Role of exonuclease III and endonuclease IV in repair of pyrimidine dimers initiated by bacteriophage T4 pyrimidine dimer-DNA glycosylase.

The role of exonuclease III and endonuclease IV in the repair of pyrimidine dimers in bacteriophage T4-infected Escherichia coli was examined. UV-irradiated T4 showed reduced survival when plated on an xth nfo double mutant but showed wild-type survival on either single mutant. T4 denV phage were equally sensitive when plated on wild-type E. coli or an xth nfo double mutant, suggesting that these endonucleases function in the same repair pathway as T4 pyrimidine dimer-DNA glycosylase. A uvrA mutant of E. coli in which the repair of pyrimidine dimers was dependent on the T4 denV gene carried on a plasmid was constructed. Neither an xth nor an nfo derivative of this strain was more sensitive than the parental strain to UV irradiation. We were unable to construct a uvrA xth nfo triple mutant. In addition, T4, which turns off the host UvrABC excision nuclease, showed reduced plating efficiency on an xth nfo double mutant.

Apurinic Acid↗
PubMed1988

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↗
PubMed1988

IST2: an insertion sequence from Thiobacillus ferrooxidans.

The genome of Thiobacillus ferrooxidans (strain ATCC 19859) contains at least two families of repeated sequences, termed family 1 and 2. The nucleotide sequence of a family 2 member was determined. It is 1408 base pairs long and has structural features similar to those of insertion sequences (IS elements). Terminal inverted repeats 25 base pairs in length are present. These inverted repeats are imperfect and adjacent to target-site duplications 9 base pairs in length. Several open reading frames were detected (the longest was 888 base pairs). We have named this IS element-like sequence IST2. The ends of a second example of IST2 were analyzed and compared to those of the first. The DNA sequences are identical and similarly sized target-site duplications are present.

Base Sequence↗
PubMed1988

T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs.

We have formed four-arm branched DNA junctions that contain no more than a single base pair of branch migratory freedom. Recently, we have shown that these Holliday junction analogs have twofold symmetric protection patterns in solution when probed with hydroxyl radicals: two opposite strands of one junction show extensive protection near the branch point, while the other pair of opposite strands is virtually as susceptible as a double helix. In a different junction, the hydroxyl radical protection pattern is reversed. These patterns suggest that a crossover-isomer bias exists in these molecules and that the protected strands form the crossover between helices. Here, we examine the cleavage pattern of these structures when they are resolved by T4 endonuclease VII. Junctions are formed from a single shamrock-shaped molecule, which contains 5', 3', or internal labels. The enzyme shows a preference for resolving these modified junctions at sites near those protected from hydroxyl radicals. This result suggests that only crossover strands in a Holliday junction are cleaved, and thus an odd number of crossover isomerizations must occur when flanking markers are exchanged.

Base Sequence↗