Faculty Bibliography

Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20x the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein

This paper describes a reductive amination crosslinking protocol that facilitates identification and characterization of a class of DNA repair enzymes, DNA glycosylase/AP lyases, which are involved in base excision repair. This crosslinking technique has been used to identify enzymes in crude extracts and in partially purified enzyme preparations, to isolate proteins for sequencing, and to confirm the reaction mechanism of members of this enzyme family. Chemical reduction of the Schiff's base enzyme-substrate intermediate to a stable amine results in the formation of an irreversible covalent bond between the substrate lesion situated within a 2'-deoxyoligonucleotide and the repair enzyme. This complex can be detected by gel electrophoresis and can also be isolated and analyzed by amino acid sequencing

The lack of a phenotypic alteration of 5-hydroxymethyluracil (hmUra) DNA glycosylase (hmUDG) deficient Chinese hamster V79mut1 cells exposed to DNA-damaging agents known to produce hmUra has raised the question whether there might be DNA substrates other than hmUra for hmUDG. Based on the structural similarity between 5-chlorouracil (ClUra) and hmUra and the observations that 5-chloro-2'-deoxyuridine (CldUrd) induces base excision repair (BER) events, we asked whether hmUDG or some other DNA BER enzyme is responsible for the removal of ClUra from DNA. An in vivo flow cytometry assay with FITC-anti-BrdUrd (which cross-reacts with CldUrd) showed that exogenous CldUrd is incorporated into DNA. However, both in vivo and in vitro experiments indicated that ClUra is not excised from DNA by hmUDG or other DNA glycosylase activities. The absence of removal of ClUra by hmUDG raised the question whether DNA strand breaks occurred subsequent to thymidylate synthase inhibition, leading to deoxyuridine incorporation, followed by cleavage of uracil from DNA by uracil DNA glycosylase (UDG). An in vivo thymidylate synthase activity assay in V79 cells demonstrated that CldUrd treatment inhibits thymidylate synthase as effectively as 5-fluoro-2'-deoxyuridine (FdUrd) treatment. Uracil, a known UDG inhibitor, partially reverses the cytotoxic effects of CldUrd on V79 cells, thus confirming that CldUrd induced cytotoxicity is a result of UDG activity. Our results demonstrated that while CldUrd is not directly repaired from DNA, its cytotoxicity is directly due to the UDG removing uracil subsequent to inhibition of thymidylate synthase by CldUMP

We previously purified a bovine pyrimidine hydrate-thymine glycol DNA glycosylase/AP lyase. The amino acid sequence of tryptic bovine peptides was homologous to Escherichia coli endonuclease III, theoretical proteins of Saccharomyces cerevisiae and Caenorhabditis elegans, and the translated sequences of rat and human 3'-expressed sequence tags (3'-ESTs) (Hilbert, T. P., Boorstein, R. J., Kung, H. C., Bolton, P. H., Xing, D., Cunningham, R. P., Teebor, G. W. (1996) Biochemistry 35, 2505-2511). Now the human 3'-EST was used to isolate the cDNA clone encoding the human enzyme, which, when expressed as a GST-fusion protein, demonstrated thymine glycol-DNA glycosylase activity and, after incubation with NaCNBH3, became irreversibly cross-linked to a thymine glycol-containing oligodeoxynucleotide, a reaction characteristic of DNA glycosylase/AP lyases. Amino acids within the active site, DNA binding domains, and [4Fe-4S] cluster of endonuclease III are conserved in the human enzyme. The gene for the human enzyme was localized to chromosome 16p13.2-.3. Genomic sequences encoding putative endonuclease III homologues are present in bacteria, archeons, and eukaryotes. The ubiquitous distribution of endonuclease III-like proteins suggests that the 5,6-double bond of pyrimidines is subject to oxidation, reduction, and/or hydration in the DNA of organisms of all biologic domains and that the resulting modified pyrimidines are deleterious to the organism

We previously demonstrated that the primary endonuclease III sensitive sites formed in DNA by UV irradiation are cytosine hydrates which are released from the polymer by the DNA glycosylase activity of the repair enzyme. The enzymatic release of uracil hydrate. the deamination product of 6-hydroxy-5,6-dihydrocytosine was also shown (1,2). Ganguly et al [3] subsequently reported that the endonuclease ill-mediated release of thymine hydrate from UV-irradiated poly(dA-(H-3)dT) In light of these results. we asked whether UV irradiation may cause hydration of 5-methylcytosine and whether such a hydrate underwent deamination to 6-hydroxy-5,6-dihydrothymine. For this purpose we irradiated an alternating copolymer containing 5-methylcytosine, (H-3-methyl)poly(dG-mdC). poly(dG-mdC) and incubated it with endonuclease III. It should be noted that no 5-methycytosine hydrate was released. Instead we observed that non-enzymatically mediated release of both intact 5-methylcytosine and 5-methylcytosine derived material from irradiated DNA. We then irradiated poly(dA-(H-3)dT) with endonuclease III in an attempt to detect enzymatically mediated release of thymine hydrate as has been reported. Instead of formation of 6-hydroxy-5,6-dihydrothymine, we observed the direct release of free thymine and thymine derived material similar to that seen with 5-methylcytsine polymer. Such non-enzymatically mediated release was found to be oxygen dependent. In contrast, the photohydration of cytosine was oxygen independent. The UV irradiation of oxygenated cells may result in fragmentation and release of 5-methylcytosine from DNA giving rise to apyrimidinic (AP) sites. The persistence of such AP sites may explain the UV sensitivity of E. coli nthnfo mutants which lack AP endonuclease activity

Our previous studies have demonstrated that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from the enzymatic removal of large numbers of hmUra residues (MCB 12:5536, 1992). Exposure of V79 cells to hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry, and light microscopy. At least 25% of the DNA repair dependent hmdUrd induced cell death in V79 cells is apoptosis. Pulse field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2 Mb - 4.5 Mb) DNA double strand breaks prior to internucleosomal fragmentation in V79 cells. HmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in base excision repair of hmUra. The apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis is not the direct consequence of NAD depletion. Our results indicate that the base excision repair process leads to the generation of high molecular weight DNA breaks, resulting in apoptosis in V79 cells. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that repair induced apoptosis is V79 cells is independent of normal p53 function. Our results indicate that apoptosis following DNA damage may be a consequence of DNA repair activity

We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny

Exposure of pyrimidines of DNA to ionizing radiation under aerobic conditions or oxidizing agents results in attack on the 5,6 double bond of the pyrimidine ring or on the exocyclic 5-methyl group. The primary product of oxidation of the 5,6 double bond of thymine is thymine glycol, while oxidation of the 5-methyl group yields 5-hydroxymethyluracil. Oxidation of the 5,6 double bond of cytosine yields cytosine glycol, which decomposes to 5-hydroxycytosine, 5-hydroxyuracil and uracil glycol, all of which are repaired in DNA by Escherichia coli endonuclease III. We now describe the products of oxidation of 5-methylcytosine in DNA. Poly(dG-[3H]dmC) was gamma-irradiated or oxidized with hydrogen peroxide in the presence of Fe3+ and ascorbic acid. The oxidized co-polymer was incubated with endonuclease III or 5-hydroxymethyluracil-DNA glycosylase, to determine whether repairable products were formed, or digested to 2'-deoxyribonucleosides, to determine the total complement of oxidative products. Oxidative attack on 5-methylcytosine resulted primarily in formation of thymine glycol. The radiogenic yield of thymine glycol in poly(dG-dmC) was the same as that in poly(dA-dT), demonstrating that 5-methylcytosine residues in DNA were equally susceptible to radiation-induced oxidation as were thymine residues

We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5-methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions

5-Methylcytosine is a minor component of the DNA of mammals and other higher eukaryotic organisms. It appears to play an important role in the control of gene expression and in other physiological processes. Both somatic mutations and germ line mutations appear to take place preferentially at sites of 5-methylcytosine residues. We have been studying the products formed from methylcytosine residues which can be produced by radiation and by oxidative stresses. We have also been studying the pathways by which such products can be repaired. Three classes of modifications to methylcytosine are being characterized: (i) lesions resulting from oxidation of the methyl group, (ii) lesions resulting from deamination, and (iii) lesions resulting from photohydration of the 5,6-ethylenic bond. Several interrelated base excision repair pathways appear to have developed to respond to these lesions. Our current studies are designed to evaluate the relative importance of these lesions and the corresponding repair pathways in mammalian cells