Biomedical subjects
Publications and source records attributed to D E Helland.
Using Southern blot analysis of DNA from a panel of rodent-human somatic cell hybrids with known karyotypes, we have assigned the human uracil-DNA glycosylase gene to chromosome 12.
The development of new methods and techniques in biotechnology over the last two decades has opened the way for fresh strategies to develop new and better vaccines. The relevant techniques have been developed through important discoveries in immunology, gene technology and polymer chemistry. The purpose of this short review is to discuss these techniques in the light of vaccine development, without going into details regarding each specific disease and vaccine.
DNA photosensitization by several furocoumarins (including 3-carbethoxypsoralen (3-CPs), 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP) and angelicin was investigated by using DNA sequencing methodology. 3-CPs induces photo-oxidation of guanine residues leading to alkali-labile sites in DNA (revealed by hot piperidine), whereas 8-MOP, 5-MOP and angelicin do not. There is a preferential photo-oxidation of G when located on the 5' side of GG doublets, likely to reflect a better accessibility of the G moiety in such a context. Mechanisms operating via both radicals (type I) and singlet oxygen (type II) are involved in the photo-oxidation of G residues by 3-CPs. Photo-oxidized G residues are produced independently of the formation of photoadducts, and scavengers of singlet oxygen or radicals do not inhibit photobinding of 3-CPs to DNA. This leads us to propose that covalent photoadducts arise from the intercalated excited sensitizer molecules, whereas G photo-oxidations are produced either by electron transfer reactions involving bound 3-CPs or by energy transfer to molecular oxygen, thereby producing singlet oxygen that subsequently reacts with guanine bases. Quantification of both types of DNA lesions indicated that in vitro photo-oxidized G residues are produced in DNA by 3-CPs plus ultraviolet light at least to the same extent as photoadducts, under our conditions. A calf thymus redoxyendonuclease, equivalent to the endonuclease III of Escherichia coli, specific for oxidative DNA damages, recognizes and cleaves DNA at sites of photo-oxidized G residues. The extent of the cleavage by this enzyme was close to that observed by hot piperidine and followed the amount of photo-oxidized G residues produced when the lifetime of excited oxygen species is modified. The redoxyendonuclease did not incise DNA treated with 8-MOP, 5-MOP or angelicin plus ultraviolet light. The exonuclease III and endonuclease IV of E. coli also involved in the repair of oxidative DNA damage, convert the replicative form I of 3-CPs-treated DNA to replicative form II. This suggests that the lesions recognized by these enzymes are apurinic-like lesions. In view of the low toxicity and mutagenicity of 3-CPs, DNA photo-oxidation products induced by the photodynamic effect of 3-CPs are likely to be efficiently taken care of by the DNA repair system(s). It is clear that 3-CPs photo-induces several classes of DNA damage, including oxidative damage.(ABSTRACT TRUNCATED AT 400 WORDS)
An apparently homogeneous enzyme preparation, containing an apurinic/apyrimidinic (AP) endonuclease from human placenta, was by DNA sequencing analysis found to act as a class I AP-endonuclease, i.e. produce a 3'-deoxyribose and 5'-phosphomonoester nucleotide termini.
An apurinic/apyrimidinic (AP) specific endonuclease from mouse plasmacytoma cells (line MPC-11), was observed to cleave apurinic sites in oligonucleotides 9, 11, 12, 39 and 40 nucleotides in length. However, the enzyme failed to cleave AP-sites in two oligonucleotides 7 nucleotides in length. The maximum rates of digestion observed on these short single-stranded DNA (ssDNA) fragments were approximately 1/30 of the rates observed on double-stranded DNA (dsDNA). In studies using the Maxam-Gilbert DNA sequencing analysis, apurinic sites in purine-rich regions were preferentially cleaved in dsDNA but not in ssDNA, indicating that the enzyme has a sequence preference on dsDNA. These results suggest that some sites on DNA might be more efficiently repaired than others.
Uracil-DNA glycosylase is the DNA repair enzyme responsible for the removal of uracil from DNA, and it is present in all organisms investigated. Here we report on the cloning and sequencing of a cDNA encoding the human uracil-DNA glycosylase. The sequences of uracil-DNA glycosylases from yeast, Escherichia coli, herpes simplex virus type 1 and 2, and homologous genes from varicella-zoster and Epstein-Barr viruses are known. It is shown in this report that the predicted amino acid sequence of the human uracil-DNA glycosylase shows a striking similarity to the other uracil-DNA glycosylases, ranging from 40.3 to 55.7% identical residues. The proteins of human and bacterial origin were unexpectedly found to be most closely related, 73.3% similarity when conservative amino acid substitutions were included. The similarity between the different uracil-DNA glycosylase genes is confined to several discrete boxes. These findings strongly indicate that uracil-DNA glycosylases from phylogenetically distant species are highly conserved.
We have developed a strategy by which the nature of phosphodiester bond breaks produced by various DNA-repair endonucleases and also other nucleases, can be characterized. A purified apurinic/apyrimidinic (AP) specific endonuclease from a permanently established mouse plasmacytoma cell-line (MPC-11) has been examined with respect to the exact incision site generated at the baseless site. By the aid of enzymatic treatment with calf intestinal phosphatase, the 3'-phosphatase activity of T4-polynucleotide kinase, chemical modification with piperidine in addition to the Maxam-Gilbert sequencing procedure, followed by separation on a DNA-sequencing gel, the nature of the cleaved phosphodiester bond, both 3' and 5' to the cleavage site, has been established. The AP-specific endonuclease investigated was classified as a class II AP-endonuclease according to the four possible classes of AP-endonuclease with respect to the termini produced. By use of this technique each single damaged and cleaved site can be investigated separately.
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.
3-Methyladenine-DNA glycosylase from calf thymus recognizes both 3-methyladenine (3-mAde), 7-methylguanine (7-mGua) and 3-methylguanine (3-mGua) residues in calf thymus DNA; the rate of release of 3-mAde is approximately eightfold higher than that for 7-mGua. The best DNA polymer substrates appeared to be those having an A-type helical conformation such as d(A-T)n and d(G-C)n. The Km values for release of 3-mAde and 7-mGua were approximately the same for the above mentioned two substrates whereas the Vmax for excision of 3-mAde was threefold higher than that of 7-mGua. The rate of hydrolysis of 7-mGua residues in d(G-C)n was similar to that found for the excision of 3-mAde in calf thymus DNA. The polymer d(G)n X d(C)m, which possesses a B-type helical conformation, was a poor substrate and the rate of excision here was approximately the same as with calf thymus DNA having the B-type structure. Polyamines greatly influenced the activity and at low concentrations a 50-100% increase in the release of 7-mGua, but not 3-mAde, was observed. With higher concentrations the rate of excision of both bases decreased sharply. The sequence specificity of the DNA glycosylase on naturally occurring DNA was studied using methylated DNA fragments from the plasmid pUC18. The results revealed that some 3-mAde as well as 7-mGua residues were seldom attacked. These 3-mAde residues were positioned either 5' to another Ade residue or in a stretch of pyrimidines, and the 7-mGua residue 3' to another Gua residue. The 3-mAde residue most frequently recognized was situated 3' to another Ade residue, and in the case of 7-mGua it was the central Gua residue in the sequence -G-G-G-.
A DNA endonuclease activity present in calf thymus specific for incision on DNA damaged by ultraviolet light, osmium tetroxide, potassium permanganate, hydrogen peroxide and acid has been purified from whole cell extracts. The enzymatic activity was heterogeneous both with regard to molecular mass and charge. The molecular mass of the enzyme varied from 25 to 35 kDa, but the different enzymatic species appeared to possess similar activities. The enzymes acted equally well on damage in supercoiled and relaxed forms of DNA. It further had a narrow optimum with regard to salt concentrations, the optimum activity being observed at a concentration of KCl from 40 to 65 mM.
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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.
3-Methyladenine-DNA glycosylase activities have been identified in all eukaryotic cell systems studied. Some of the results from these studies are reviewed here. The enzymes possess molecular weights between 24 X 10(3) and 34 X 10(3), they have a broad pH optimum at approximately pH 8, require double-stranded DNA and act in the absence of any cofactors. The enzyme can excise several different methylated bases from DNA such as 3-methyladenine, 7-methylguanine and 3-methylguanine. The specific activity of this DNA glycosylase in mouse L-cells was found to be a function of the proliferative state of the cell. In vitro quantification of this DNA repair activity in synchronized mouse L-cells suggests that it is regulated within a defined temporal sequence prior to the onset of DNA replication. Using DNA fragments of defined sequences it was observed that the efficiency of removal of the methylated bases is sequence-dependent.
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.
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.
The properties of a DNA-repair endonuclease isolated from mouse plasmacytoma cells have been further studied. It acted on ultraviolet-light-irradiated supercoiled DNA, and the requirement for a supercoiled substrate was absolute at ultraviolet light doses below 1.5 kJ m-2. At higher doses relaxed DNA could also serve as a substrate, but the activity on this DNA was due mostly to hydrolysis of ultraviolet-light-induced apurinic/apyrimidinic (AP) sites by the AP-endonuclease activity associated with the enzyme. The latter enzyme activity did not require a supercoiled form of the DNA. The enzyme also introduced nicks in unirradiated d(A-T)n. The nicked ultraviolet-light-irradiated DNA served as a substrate for DNA polymerase I, showing that the nicks contained free 3'-OH ends. Treatment of the nicked ultraviolet-light-irradiated DNA with bacterial alkaline phosphatase followed by T4 polynucleotide kinase, resulted in the phosphorylation of the 5' ends of the nicks, indicating that the nicks possessed a 5'-phosphate group; 5'- and 3'-mononucleotide analyses of the labelled DNA suggested that the enzyme introduced breaks primarily between G and T residues. The enzyme did not act on any specific region on the supercoiled DNA molecule; it produced random nicks in ultraviolet-light-modified phi X 174 replicative form I DNA. Antibodies raised against ultraviolet-light-irradiated DNA inhibited the activity. DNA adducts such as N-acetoxy-2-acetylaminofluorene and psoralen were not recognized by the enzyme. It is suggested that the enzyme has a specificity directed toward helical distortions.
The 3-methyladenine-DNA glycosylase from calf thymus has been purified and characterized. Two species of Mr = 42,000 and 27,000 +/- 5% and Stokes radius of 27.5 and 22.4 A, respectively, were found. Only the lower molecular weight species were present in the nucleus; it was bound to chromatin and could be dissociated in the presence of 0.25 M KCl. The enzymatic properties of the two species appeared to be identical. Both enzyme species released 3-methyladenine, 7-methylguanine, and 3-methylguanine, listed in the order of decreasing activity. The chromatin-associated enzyme was purified to apparent homogeneity and found to be a basic protein having a pI greater than 9. It was completely inhibited by p-hydroxymercuribenzoate, but this inhibition could be fully reversed by addition of excess 2-mercaptoethanol. Kinetic studies, heat inactivation, and inhibition experiments demonstrated that the 3-methyladenine and 7-methylguanine releasing activities were located on the same protein molecule. The enzymes showed no activity on methylated single-stranded DNA. No product inhibition was observed for any of the enzyme species, and the enzyme activity was optimal when the incubation was performed in the presence of 50 mM NaCl or KCl at pH values between 8 and 9.
Escherichia coli B exposed to high doses of bacteriophage T7 did not lyse. A similar effect was observed when the high dose was added within the first 7 min after primary infection. No viable phage was formed. DNA synthesis was inhibited rapidly and the nucleoid structure was absent. Protein synthesis was in general markedly reduced and so were the activities of the phage-specific enzymes endolysin and DNA polymerase. However, phage genes were transcribed both by the host RNA polymerase and by the phage-specific enzyme. We suggest that inhibition of phage development is due to structural alterations occurring in the cell wall/membrane such that replication is inhibited.