Substrate specificity of the Escherichia coli endonuclease III: Excision of thymine- and cytosine-derived lesions in DNA produced by radiation-generated free radicals (original) (raw)
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Journal of Biological Chemistry, 1988
Agents that act via oxygen-derived free radicals form DNA strand breaks with fragmented sugar residues that block DNA repair synthesis. Using a synthetic DNA substrate with a single type of sugar fragment, 3'-phosphoglycolaldehyde esters, we show that in Escherichia coli extracts the only EDTA-resistant diesterase for these damages depends on the bacterial nfo (endonuclease IV) gene. Endonuclease IV was purified to physical homogeneity (Mr = 31,000) from an E. coli strain carrying the cloned nfo gene and in which the enzyme had been induced with paraquat. Although heat-stable and routinely assayed in the presence of EDTA, endonuclease IV was inactivated in the absence of substrate at 23-50 "C by either EDTA or 1,lOphenanthroline, suggesting the presence of an essential metal tightly bound to the protein. Purified endonuclease IV released phosphoglycolaldehyde, phosphate, and intact deoxyribose 5-phosphate from the 3'-end of DNA, all with apparent K, of 5-10 nM. The optimal KC1 or NaCl concentration for 3'-phosphoglycolaldehyde release was 50-100 mM. The purified enzyme had endonuclease activity against partially depurinated DNA but lacked significant nonspecific nuclease activities. Endonuclease IV also activated H,Oz-damaged DNA for repair synthesis by DNA polymerase I. Thus, endonuclease IV can act on a variety of oxidative damages in DNA, consistent with a role for the enzyme in combating free-radical toxicity. Partially reduced oxygen species are toxic and mutagenic to cells (Halliwell, 1987). These species, notably superoxide radical anion (O;), hydrogen peroxide, and hydroxyl radical (HO), are formed by ionizing radiation (Hutchinson, 1985), antibiotics such as paraquat and streptonigrin (Hassett et al., 1987), and by normal cellular metabolism (Fridovich, 1978). Living cells have a front-line enzymatic defense system against damage by oxygen radicals in the form of superoxide dismutase and catalase-hydroperoxidase (Fridovich, 1978). When these scavenging enzymes are missing (Farr et al., 1986) or noninducible (Storz et al., 1987), the spontaneous mutation frequency in Escherichia coli and Salmonella typhimurium is elevated during aerobic growth, while overproduction of per-* This work was supported by Grants CA37831 and ES03926 from the National Institutes of Health (to B. D.
Journal of Biological Chemistry, 1984
A DNA glycosylase activity that excises oxidized, fragmented thymine residues from a polydeoxyribonucleotide has been purified 9,500-fold to apparent homogeneity from Escherichia coli. The purified enzyme also excises thymine glycol and cleaves DNA at apurinic sites, and appears to be identical with E. coli DNA endonuclease 111. The enzyme catalyzes the release of several different forms of oxidized thymine, including urea, methyltartronylurea and 5-hydroxy-5-methylhydantoin. The molecular weight of the native protein is 25,000, and the same value is obtained for the denatured homogeneous protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cellular DNA continuously endures the introduction of premutagenic alterations by heat-induced hydrolysis (1) and nonenzymatic alkylation with S-adenosylmethionine (2, 3). The major structural lesions generated in this way are effectively corrected by various DNA repair processes. Normal oxygen metabolism is also associated with potentially mutagenic side effects, because of the chance occurrence of reactive ' The abbreviations used are: Hepes, N-2-hydroxyethylpiperazine
Substrate specificity of a mammalian DNA repair endonuclease that recognizes oxidative base damage
Molecular and Cellular Biology, 1986
The substrate specificity of a calf thymus endonuclease on DNA damaged by UV light, 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 wa? 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 ofEscherichia coli endonuclease HI. 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.
Toxicology Letters, 1993
A number of repair endonucleases, viz. endonuclease III, fo~amidopyrimidin~DNA glycosylase (FPG protein). endonuclease IV, exonuciease III and UV endonuclease, is used to simultaneously quantify various types of DNA modifications, which were induced by agents that generate reactive oxygen species. Under cell-free conditions, two types of DNA damage profiles are obtained. The profiles induced by chemically generated singlet oxygen and by various photosensitizers (acridine orange, methylene blue. riboflavin, hematoporphyrin) plus light are dominated by base modifications sensitive to FPG protein, while 5,6-dihydropyrimidines (recognized by endonuclease III), sites of base loss (AP sites, recognized by endonuclease IV and exonuclease III) and strand breaks are minor lesions. In contrast, the DNA damage profile induced by hydroxyl radicals (y-rays) consists of approx. equal levels of base modifications, AP sites and strand breaks. The damage profiles induced by Fe(III)-EDTA in the presence of superoxide and by Fe~III)-nitrilotri~etate in the presence of HtOz do not differ from that by hydroxyl radicals. The damage profile induced by Cu(II)-phenanthroline deviates by high levels of AP sites that are recognized by endonuclease IV and exonuclease III-but not by those AP endonucleases which cfeave at the 3' site-and probably represent AP sites oxidized at C-l'. The damage induced by Fe(III)-bleomycin plus Hz@ deviates by an increased level of double strand breaks and the absence of endonuclease-sensitive base modifications. Cellular DNA damage profiles are obtained from bacteria, cultured mammalian cells and mammalian mitochondria after exposure to acridine orange plus visible light. A comparison with the cell-free profiles reveals that the damage in all three systems is not induced indirectly by hydroxyl radicals or an activation of cellular n&eases, but by the same mechanism that is responsible for the cell-free DNA damage.
Mutation Research/DNA Repair, 1996
We report on a novel activity of T4 endonuclease V. This enzyme is well known to be specific for the excision of pyrimidine dimers from UV-irradiated DNA. In this work, we show that T4 endonuclease V excises 4.6-diamino-5-formamidopyrimidine from DNA. 4,6-Diamino-5-formamidopyrimidine is formed as a product of adenine in DNA upon action of hydroxyl radicals and upon UV-irradiation. DNA substrates were prepared by UV-or y-irradiation of DNA in aqueous solution. DNA substrates were incubated either with active T4 endonuclease V or with heat-inactivated T4 endonuclease V or without the enzyme. After incubation, DNA was precipitated and supernatant fractions were separated. Supernatant fractions after derivatization, and pellets after hydrolysis and derivatization were analyzed by gas chromatography/isotopedilution mass spectrometry. The results provide evidence for the excision of 4,6-diamino-5-formamidopyrimidine by T4 endonuclease V from both y-and UV-irradiated DNA. Kinetics of excision were also determined. Fifteen other pyrimidineand purine-derived base lesions that were identified in DNA samples were not substrates for this enzyme. It was concluded that, in addition to its well known activity for pyrimidine photodimers, T4 endonuclease V possesses an N-glycosylase activity for a major UV-radiation-and hydroxyl radical-induced monomeric product in DNA.
5,6-Saturated thymine lesions in DNA: production by ultraviolet light or hydrogen peroxide
Nucleic Acids Research, 1982
Thymine analogs with saturated 5-6 bonds are important types of DNA damage that are recognized by the DNA N-glycosylase activity of E. coli endonuclease III. Seeking agents which could preferentially form 5,6-hydrated thymine residues in duplex DNA both in vivo and in vitro, we exposed purified duplex DNA to 325or 313-nm light; however, after such exposure pyrimidine diners greatly predominated over 5,6-hydrated thymine. Hydrogen peroxide, on the other hand, formed significant numbers of endonuclease III-sensitive sites in vitro which were not apurinic/apyrimidinic lesions and thus were likely to be 5,6-hydrated thymines.
Multiply damaged sites in DNA: interactions with Escherichia coli endonucleases III and VIII
Nucleic Acids Research, 1998
Bursts of free radicals produced by ionization of water in close vicinity to DNA can produce clusters of opposed DNA lesions and these are termed multiply damaged sites (MDS). How MDS are processed by the Escherichia coli DNA glycosylases, endonuclease (endo) III and endo VIII, which recognize oxidized pyrimidines, is the subject of this study. Oligonucleotide substrates were constructed containing a site of pyrimidine damage or an abasic (AP) site in close proximity to a single nucleotide gap, which simulates a free radical-induced single-strand break. The gap was placed in the opposite strand 1, 3 or 6 nt 5′ or 3′ of the AP site or base lesion. Endos III and VIII were able to cleave an AP site in the MDS, no matter what the position of the opposed strand break, although cleavage at position one 5′ or 3′ was reduced compared with cleavage at positions three or six 5′ or 3′. Neither endo III nor endo VIII was able to remove the base lesion when the gap was positioned 1 nt 5′ or 3′ in the opposite strand. Cleavage of the modified pyrimidine by endo III increased as the distance increased between the base lesion and the opposed strand break. With endo VIII, however, DNA breakage at the site of the base lesion was equivalent to or less when the gap was positioned 6 nt 3′ of the lesion than when the gap was 3 nt 3′ of the lesion. Gel mobility shift analysis of the binding of endo VIII to an oligonucleotide containing a reduced AP (rAP) site in close opposition to a single nucleotide gap correlated with cleavage of MDS substrates by endo VIII. If the strand break in the MDS was replaced by an oxidized purine, 7,8-dihydro-8-oxoguanine (8-oxoG), neither endo VIII cleavage nor binding were perturbed. These data show that processing of oxidized pyrimidines by endos III and VIII was strongly influenced by the position and type of lesion in the opposite strand, which could have a significant effect on the biological outcome of the MDS lesion.