5,6-Dihydroxy-5,6-dihydrothymine (thymine glycol) is formed in DNA by reaction with oxidizing agents and as a result of ionizing and near-ultraviolet radiation. We describe a rapid purification of cis-5,6-dihydroxy-5,6-dihydrothymine and cis-5,6-dihydroxy-5,6-dihydrothymidine (cis-thymidine glycol) and their use as markers in identifying the thymine glycol moiety in oxidized DNA. Both glycols were prepared by oxidation of [14C]thymine and -thymidine with KMnO4 followed by purification on Sephadex LH-20 (LH-20). [3H]DNA was oxidized with KMnO4 and the thymidine glycol in DNA identified by enzymatic digestion of the DNA followed by cochromatography of the digest with marker [14C]thymidine glycol on LH-20. The cis conformation of the glycol was confirmed by the change in the elution pattern when borate rather than water was used as eluent. Alkaline hydrolysis of a mixture of [14C]thymine glycol and oxidized [3H]DNA followed by trichloroacetic acid precipitation and LH-20 chromatographic analysis of the neutralized supernatant yielded a complex pattern of radioactive degradation products with coincidence of one 14C marker- and one [3H]-DNA-derived peak. All applied radioactivity was recovered. This methodology should be useful in determining thymine glycol content of irradiated DNA and in elucidating the mechanism by which these altered residues are removed from cellular DNA by repair enzymes.

DNA 5-methylcytosine is a major factor in the silencing of mammalian genes; it is involved in gene expression, differentiation, embryogenesis and neoplastic transformation. A decrease in DNA 5-methylcytosine content is associated with activation of specific genes. There is much evidence indicating this to be an enzymic process, with replacement of 5-methylcytosine by cytosine. We demonstrate here enzymic release of 5-methylcytosines from DNA by a human 5-methylcytosine-DNA glycosylase activity, which affords a possible mechanism for such replacement. This activity generates promutagenic apyrimidinic sites, which can be related to the high frequency of mutations found at DNA 5-methylcytosine loci. The recovery of most released pyrimidines as thymines indicates subsequent deamination of free 5-methylcytosines by a 5-methylcytosine deaminase activity. This prevents possible recycling of 5-methylcytosine into replicative DNA synthesis via a possible 5-methyl-dCTP intermediate synthesized through the pyrimidine salvage pathway. Taken together, these findings indicate mechanisms for removal of 5-methylcytosines from DNA, hypermutability of DNA 5-methylcytosine sites, and exclusion of 5-methylcytosines from DNA during replication.

There exist numerous genetic disorders, marked by chromosome instability, that are strikingly associated with various cancers. Both the chromosomal instabilities and neoplastic outcomes are related to abnormalities of DNA metabolism, DNA repair, cell-cycle governance, or control of apoptosis. Among these diseases are ataxia telangectasia and Nijmegen breakage syndrome, with increased incidences of lymphomas. Bloom syndrome, Werner syndrome, and Rothmund-Thompson syndrome, each characterized by a DNA helicase defect, are associated with early incidences of different cancers. Other diseases combining the phenotype of chromosomal instabilities and neoplastic development are Fanconi anemia and breast cancers associated with mutant BRCA1 and BRCA2 genes. The cloning of the encoding genes and the characterization of their products have resulted in partial understanding of the pathways of cellular DNA surveillance and maintenance of genomic rectitude. The exact pathways fully linking the genetic defect mechanisms to the eventual development of various neoplasias remain to be elucidated, but progress in defining the molecular genetics of these entities suggests that many of them are disorders of DNA recombination. Each defect involves a separate protein in these complex pathways.

Photoalkylation, the ultraviolet irradiation of DNA with isopropanol and di-tert-butylperoxide, causes a variety of base alterations. These include 8-(2-hydroxy-2-propyl)guanines, 8-(2-hydroxy-2-propyl)adenines and thymine dimers. An E. coli endonuclease against photoalkylated DNA was assayed by conversion of superhelical PM2 phage DNA to the nicked form. Enzyme activities were compared between extracts of strain BW9109 (xth-), lacking exonuclease III activity, and strain BW434 (xth-,nth-), deficient in both exonuclease III and endonuclease III. The endonuclease level in the double mutant against substrate photoalkylated DNA was under 20% of the activity in the mutant lacking only exonuclease III. Irradiation of the DNA substrate in the absence of isopropanol did not affect the activity in either strain. Analysis by polyacrylamide gel electrophoresis identified the sites of DNA cleavage by purified E. coli endonuclease III as cytosines, both in DNA irradiated at biologically significant wavelengths and in photoalkylated DNA. Neither 8-(2-hydroxy-2-propyl)purines, pyrimidine dimers, uracils nor 6-4'-(pyrimidin-2'-one)pyrimidines were substrates for the enzyme.

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