Faculty Bibliography

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

Radiation and oxidation agents damage the pyrimidine bases of DNA. The resulting modified bases can be recognized by enzymes which effect the removal of base lesions, leaving an apurinic site in the DNA backbone. We have been studying three distinct classes of pyrimidine base damage: a) saturation of the 5,6-ethylenic bond to produce the corresponding glycol, b) hydration of the 5,6 double bond to produce the corresponding 6-hydroxy-5,6-dihydroderivative, and c) oxidation of the methyl group of thymine and 5-methylcytosine to produce the corresponding hydroxymethylated compound

We isolated a mutant mammalian cell line lacking activity for the DNA repair enzyme 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). The mutant was isolated through its resistance to the thymidine analog 5-hydroxymethyl-2'-deoxyuridine (HmdUrd). The mutant incorporates HmdUrd into DNA to the same extent as the parent line but, lacking the repair enzyme, does not remove it. The phenotype of the mutant demonstrates that the toxicity of HmdUrd does not result from substitution of thymine in DNA by HmUra but rather from the removal via base excision of large numbers of HmUra residues in DNA. This finding elucidates a novel mechanism of toxicity for a xenobiotic nucleoside. Furthermore, the isolation of this line supports our hypothesis that the enzymatic repairability of HmUra derives not from its formation opposite adenine via the oxidation of thymine, but rather from its formation opposite guanine as a product of the oxidation and subsequent deamination of 5-methylcytosine

5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) is a major product of the reaction of thymidine with reactive oxygen species, including those generated by ionizing radiation. Thymidine glycol exists as 2 diastereomeric pairs by virtue of the chirality of the C(5) and C(6) atoms. A simple procedure is described for synthesizing and purifying each of the diastereomeric pairs separately. After brominating thymidine, the two trans 5-bromo-6-hydroxy-5,6-dihydrothymidine (thymidine bromohydrin) C(5) diastereomers were easily separated by High Performance Liquid Chromatography. Each thymidine bromohydrin was quantitatively converted to the corresponding diastereomeric thymidine glycol pair by reflux in aqueous solution. The concentrations at equilibrium of the cis (5S,6R),(5R,6S) and trans (5S,6S),(5R,6R) forms of the thymidine glycol diastereomers were determined and were 80% cis and 20% trans for the 5S pair and 87% cis and 13% trans for the 5R pair. At equilibrium, the rate of cis-trans epimerization of the two sets of diastereomers was essentially identical. The 5S diastereomeric pair was significantly more alkali labile than the 5R pair due to the higher concentration of the 5S trans epimer at equilibrium. This differential alkali lability was also manifest when the thymine glycol moiety was present in chemically oxidized poly(dA-dT).poly(dA-dT) indicating that the chemical differences between the diastereomeric pairs are preserved in DNA. These chemical differences may affect the biological properties of this important oxidative derivative of thymine in DNA

5-hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of Thy. It is removed from DNA by HmUra-DNA glycosylase. To determine whether the replacement of Thy by HmUra is mutagenic, which might explain the repairability of HmUra, a HmUra residue was substituted for Thy in a target (amber) codon by in vitro extension of an oligonucleotide primer annealed to phi X-174am3 virion DNA. This was accomplished by synthesizing HmdUTP and using DNA polymerase to effect primer extension. E. coli spheroplasts were transfected with the HmUra-containing DNA and the yield of revertant phage determined following replication in the bacterial host. Since E. coli do not express HmUra-DNA glycosylase activity, mutagenesis could be assessed in the absence of repair. chi 2c analysis showed that replacing Thy with HmUra did not result in an increase in revertant phage. These data indicate that the oxidation of Thy to HmUra in cellular DNA probably does not result in substantial mutagenesis

UV irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates which are repaired by bacterial and mammalian DNA glycosylases (Biochemistry, 28: 6164, 1989). E. coli endonuclease III was used to quantitate the formation and stability of these hydrates in 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 Cyt 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 photoproducts, poly(dG-dC) was incubated in solution for up to 24 hours after UV irradiation. Cytosine hydrate was stable at 4-degrees-C and decayed at 25- degrees-C, 37-degrees-C and 55-degrees-C with half lives of 75, 25 and 6 hours, respectively. 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 hours at 37- degrees-C and less than one half-hour at 55-degrees-C. Uracil hydrate and uracil were also shown to be formed in irradiated poly(dG-dC). These experiments demonstrate that far-UV induced cytosine hydrate may persist in DNA for prolonged time periods and also undergoes 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

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

5-Hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of thymine. It is also the product of the deamination of 5-hydroxymethylcytosine (HmCyt) which may be formed via oxidation of 5-methylcytosine (MeCyt). HmUra is removed from DNA by a DNA glycosylase which, together with HmCyt-DNA glycosylase, is unique among DNA repair enzymes in being present in mammalian cells but absent from bacteria and yeast. We found HmUra-DNA glycosylase activity in a wide variety of vertebrate and invertebrate animals (except Drosophila) and in protozoans. In most vertebrate organisms the highest specific activity was in nervous and immune system tissue. The phylogenetic distribution of HmUra-DNA glycosylase correlates with the presence of 5-methylcytosine (MeCyt) as a regulator of gene expression. This distribution of activity supports the contention that HmUra-DNA glycosylase aids in the maintenance of methylated sites in DNA

5-Hydroxymethyluracil (HmUra) residues formed by the oxidation of thymine are removed from DNA through the action of a DNA glycosylase activity. This activity was purified over 1870-fold from calf thymus and found to be distinct from uracil (Ura)-DNA glycosylase. The HmUra-DNA glycosylase has a molecular weight of 38,000, a pH optimum of 6.7-6.8 and an apparent Km of 0.73 +/- 0.04 microM. These values are similar to those reported for other mammalian DNA glycosylases. The enzyme removed HmUra residues from single- and double-stranded DNA with almost equal efficiency. HmUra-DNA glycosylase activity was not product inhibited by free HmUra. The DNA glycosylase activity was inhibited by Mg2+, but the purest enzyme fractions contained a Mg2+-dependent apurinic/apyrimidinic endonuclease activity. HmUra-DNA glycosylase and the recently described 5-hydroxymethylcytosine (HmCyt)-DNA glycosylase (Cannon, S. V., Cummings, A. C., and Teebor, G. W. (1988) Biochem. Biophys. Res. Commun. 151, 1173-1179) are unique among known DNA glycosylases in being present in mammalian cells and absent from bacteria. These DNA glycosylase activities were shown here to reside on different proteins. We suggest that the major function of HmUra-DNA glycosylase, together with HmCyt-DNA glycosylase, is the maintenance of methylated cytosine residues in the DNA of higher organisms

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