DNA glycosylases

Caradonna, Salvatore J.; Cheng, Yung‐Chi

doi:10.1007/bf00215581

Cited by 18 publications

(13 citation statements)

References 65 publications

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“…Most derivatives modifi ed at positions 5 and 6 are also ineffective, but 6-aminouracil and 5-azauracil inhibit almost as well as unsubstituted uracil, and 5-fluorouracil is a weak inhibitor (11). These data suggest that the enzyme is able to remove the latter derivaties if incorporated into DNA, and this has already been shown in the case of 5-fl uorouracil (16)(17)(18), which is excised by either the E. coli or human uracil-DNA glycosylase, albeit 20 times more slowly than unsub stituted uracil. In addition, 5-fiuoro-dUTP is a substrate for the cellular dUTPase, so the mechanisms for avoiding incorporation of 5-fl uorouracil into DNA are almost the same as those employed to prevent uracil incorpo ration (16)(17)(18).…”

Section: Dna Glycosylasesmentioning

confidence: 66%

DNA Repair Enzymes

Lindahl¹

1982

Annu. Rev. Biochem.

857

445

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Section: Dna Glycosylasesmentioning

confidence: 66%

DNA Repair Enzymes

Lindahl¹

1982

Annu. Rev. Biochem.

857

445

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“…The modified ODNs (Table 1, X ϭ abasic site) were prepared by treating the unmodified ODNs (Table 1, where X is uracil) with uracil DNA glycosylase (UDG) to remove uracil, leaving behind an abasic site [21,22]. For T3U (Table 1, X ϭ abasic site), 15 L of 5'-d(TTUTTT) (0.65 g/L) was mixed with 2 L of 10ϫ UDG buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM EDTA).…”

Section: Methodsmentioning

confidence: 99%

“…We chose pyrimidine-rich ODNs because our long-range goal is to introduce limited numbers of purines into model ODNs and use them as reaction centers for carcinogens. The chosen ODNs were treated with uracil DNA glycosylase (UDG), which selectively excises uracil from DNA by hydrolyzing the N-glycosidic bonds between uracil and the sugar moiety, leaving behind an abasic site in which an OH replaces the nucleobase [21,22].…”

Section: Molecular Weight and Stability Determinationmentioning

confidence: 99%

“…They were prepared from chromatography beads (AG50W-X8, 100 -200 mesh; Bio-Ad, Melville, NY) in the H ϩ form by using a literature procedure [20]. The Escherichia coli uracil DNA glycosylases (UDG) was obtained from Life Technologies Inc. (Rockville, MD).The ODNs in Table 1 (X ϭ uracil) and those used as standards for MALDI calibration, [d(T5), d(T10), d(T18)] were synthesized at the Nucleic Acid Chemistry Laboratory, Washington University School of Medicine.The modified ODNs (Table 1, X ϭ abasic site) were prepared by treating the unmodified ODNs (Table 1, where X is uracil) with uracil DNA glycosylase (UDG) to remove uracil, leaving behind an abasic site [21,22]. For T3U (Table 1, X ϭ abasic site), 15 L of 5'-d(TTUTTT) (0.65 g/L) was mixed with 2 L of 10ϫ UDG buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM EDTA).…”

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confidence: 99%

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Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

Zhang

Gross

2002

J. Am. Soc. Mass Spectrom.

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We describe two approaches employing electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and matrix assisted laser desorption/ionization (MALDI) post-source decay (PSD) for determining the location of an abasic site in modified oligodeoxynucleotides (ODNs). With MS/MS, we found both complementary fragment ions (a n ' and w n ') produced at the abasic site were predominant in the mass spectra and allowed the location to be determined. Under MALDI conditions, most ODNs carrying an abasic site are singly charged, and PSD gives predominately w n ' ions at the abasic sites, revealing their location. We also describe another approach for identifying and locating abasic sites in model ODNs; namely, an "in situ" derivatization coupled with MALDI mass spectrometry (MS). In general, an ODN n-mer containing an abasic site at the m-th position from the 5'-terminus can react with the matrix component, anthranilic acid, to form a Schiff base. The adduct upon MALDI breaks into 3' and 5' fragments (w nϪm , b m , a m , d mϪ1 ) at the abasic site, revealing its location. ESI MS methods are also applicable for detecting the hydrazone derivatives of abasic sites, and the fragmentation of hydrazones shows the location of the abasic site. . Thus far, mass spectrometry has played a minor role in locating abasic sites. Two recent reports show that the use of enzymes to release small oligodeoxynucleotides followed by simple mass measurement [2,3] can be an effective approach to locating abasic sites. Others used enzymes to produce abasic sites and made use of the ease of alkaline cleavage at those sites to produce small ODNs, which can be mass-measured by matrix-assisted laser desorption ionization (MALDI) mass spectrometry [4]. Another recent development of a physical method for detecting abasic sites on DNA is atomic force microscopy [5]. Despite these advances, there remains a need to develop alternative methods that make fuller use of the capabilities of mass spectrometry and that can be used for ODNs either taken as models in studies of DNA damage or as fragments released from damaged DNA.One approach would be to implement tandem mass spectrometry, which is now emerging as an effective tool for structural analysis and quantification of modified ODNs [6 -16]. Vouros and co-workers [11], in an early example, showed that the combination of electrospray ionization (ESI) coupled with tandem mass spectrometry (MS/MS) can determine the molecular mass and the sequence of short, modified ODNs. That group later investigated the chemo selectivity of a carcinogenic diol metabolite on reaction in vitro with an ODN dodecamer [12]. We recently demonstrated that ESI MS/MS and MALDI PSD are successful strategies to distinguish both normal and UV photo-damaged ODNs containing 4 -8 nucleotides [13,14]. Vouros and coworkers [15] also reported a similar method to identify and locate an aflatoxin B 1 modified guanine in three isomeric ODN 9-mers. The latter work of Vouros [15] and that of Sheil's groups [16] demonstrated the simple and inform...

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“…This enhancement of repair capacity has been observed with both asynchronous and synchronous cell populations, using various protocols to quantitate DNA repair. The in vitro quantitation of individual DNA repair enzyme activities during cell proliferation demonstrated that increases in the specific activities of the base excision repair enzymes uracil DNA glycosylase (8)(9)(10)(11)(12)(13)) and 3-methyladenine DNA glycosylase (12) as well as an increase in the specific activity of the 06-methylguanine methyltransferase (14) were dependent on the proliferative state of the cell. These increases in enzyme activity, in the absence of cellular insult, indicated that the induction of repair enzymes was a normal regulatory event during cell proliferation (15,16).…”

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confidence: 99%

Isolation and characterization of monoclonal antibodies directed against the DNA repair enzyme uracil DNA glycosylase from human placenta.

Arenaz¹,

Sirover²

1983

Proc. Natl. Acad. Sci. U.S.A.

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A series of monoclonal antibodies has been prepared against the base excision repair enzyme uracit DNA glycosylase isolated from human placenta. Spleen cells from BALB/ c mice immunized with purified human placentaluracil DNA glycosylase were fused with either P3X63 Ag8.653 or SP2/0 myeloma cells. Hybridomas producing antibodies directed against the placental glycosylase were identified in an enzyme-linked immunosorbent assay. Each positive hybridoma was cloned twice by limit dilution and tested for anti-glycosylase activity in an enzyme immunoprecipitation assay. Each of the four clones examined in detail precipitated enzyme activity in an immunoprecipitation reaction only in the presence of rabbit anti-mouse IgG as a second antibody. No anti-uracil DNA glycosylase activity was observed in a spontaneous hybridoma used as a control. Each monoclonal antibody immunoprecipitated uracil DNA glycosylases isolated from several human tissues. Partial crossreactivity was observed with rat liver glycosylase and with a hamster enzyme. In contrast, no crossreactivity was observed with yeast or Escherichia coli glycosylase. Glycerol gradient sedimentation analysis demonstrated that one of the antibodies bound to the glycosylase at a site that did not diminish its catalytic activity. A second monoclonal antibody bound at a determinant that affected catalytic activity. Analysis of antibody-glycosylase interactions suggests that human cells contain antigenically distinct glycosylase species that may be encoded by individual uracil DNA glycosylase genes. The potential use of these monoclonal antibodies in studies examining the regulation of.glycosylase isoenzymes during cell proliferation in normal human cells and in cells from cancer-prone individuals is considered.Recent studies have indicated that there is a correlation between the proliferative state of a eukarvotic cell and the capacity of that cell to repair DNA after chemical or physical insult. Increases in the.repair capacity of proliferating cells above basal levels present in quiescent cells have been observed for all DNA repair pathways that have been examined. These have included nueleotide excision repair after UV irradiation (1-6) or after exposure to N-acetoxyacetylaminofluorene (2, 6), base excision repair after exposure to methyl methanesulfonate (5, 6) or to sodium bisulfite (5), and repair after exposure to ionizing radiation (7). This enhancement of repair capacity has been observed with both asynchronous and synchronous cell populations, using various protocols to quantitate DNA repair. The in vitro quantitation of individual DNA repair enzyme activities during cell proliferation demonstrated that increases in the spe-

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DNA glycosylases

Cited by 18 publications

References 65 publications

DNA Repair Enzymes

DNA Repair Enzymes

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

Isolation and characterization of monoclonal antibodies directed against the DNA repair enzyme uracil DNA glycosylase from human placenta.

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