Detection of UV purine photoproducts in a defined sequence of human DNA.

The UV-irradiated, 3'-end-labeled, 92-base-pair terminus of the human alphoid sequence was incubated with purified endonuclease v. Previously unreported photoproducts were incised at purine loci. These were not pyrimidine photodimers, 6-4'-(pyrimidin-2'-one)-pyrimidines, base loss sites, or ring-opened purines. Therefore, purine-containing photoproducts, possibly dimers, were incised by the enzyme preparation.

Inhibition of enzymic incision of thymine dimers by covalently bound guanine adducts of 4-nitroquinoline-1-oxide in DNA.

The effects of the presence in DNA of covalently bound guanine adducts of the carcinogen 4-nitroquinoline-1-oxide on the pyrimidine dimer-DNA glycosylase, purified from bacteriophage T4-infected Escherichia coli, were investigated. E. coli DNA, labeled in thymine, photosensitized by silver nitrate, and irradiated by 254 nm monochromatic light, was the substrate. 4-Nitroquinoline-1-oxide was reduced to 4-hydroxyaminoquinoline-1-oxide and then reacted with irradiated DNA in the presence of seryl-AMP, yielding covalently bound adducts in DNA. These were assayed by high performance liquid chromatography. Enzyme activity was assayed by measuring release of labeled free thymine from directly photoreversed DNA after the reaction. Glycosylase activity was reduced against carcinogen-modified DNA, with the Vmax 38% of that against the control DNA; the Km was unaffected. Therefore, as with other modified purines, 4-nitroquinoline-1-oxide guanine modifications can reduce enzymic incision at thymine dimers. Left unrepaired, pyrimidine dimers are both mutagenic and carcinogenic. This is consistent with the possibility that interference with enzymic initiation of DNA excision repair of UV damage may be an indirect mechanism of mutagenesis by stable carcinogen-DNA adducts.

Perturbations of enzymic uracil excision due to guanine modifications in DNA.

Phage PBS2 DNA, which contains uracil in place of thymine, was used as substrate for purified Bacillus subtilis uracil:DNA glycosylase. Incubation of this DNA with the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene resulted in the production of N-(deoxyguanosin-8-yl)acetylaminofluorene. A decreased Vmax resulted from the reaction of the glycosylase with this arylamidated substrate. Addition of a 2-fold excess of control PBS2 DNA following initiation of the reaction with the modified substrate showed delayed dissociation of the enzyme from the arylamidated DNA. This shows that the presence of a carcinogen-modified DNA base can reduce the capacity for uracil excision. Therefore, interference with enzymic release of uracil from DNA may be an indirect mechanism of mutagenesis by carcinogen:DNA adducts.

Partial purification and characterization of human 5-methylcytosine-DNA glycosylase.

The molecular mechanisms by which DNA 5-methylcytosine content is modulated are incompletely understood. Reduction of DNA 5-methylcytosine content has been correlated with the transition from hyperplasia to adenoma in the genesis of human adenocarcinoma of the colon. 5-methylcytosine-DNA glycosylase removes 5-methylcytosine from DNA as a free base, but its involvement in this process is unknown. The 5-methylcytosine-DNA glycosylase activity in HeLa nuclear extracts has been partially purified, with a 460-fold enrichment, and characterized. This activity is specific for 5-methylcytosine at CpG sites in fully methylated DNA; hemimethylated DNA is not a significant substrate. DNA containing unmethylated cytosines is not cleaved by the enzyme. There is an absolute requirement for Mg2+ ions for the activity, which is inhibited by EDTA. This 5-methylcytosine-DNA glycosylase activity could be involved in carcinogenesis, transcription, replication, differentiation and development through resultant DNA hypomethylation following enzymatic removal of 5-methylcytosine from DNA.

Normal endonuclease activities for damaged DNA during hepatocarcinogenesis.

Two endonuclease activities in rat liver for damaged DNA were assayed. Double-stranded, covalently closed DNA from phage PM2 was damaged by either ultraviolet irradiation or by heating at acid pH, and used as substrate for endonucleases specific for ultraviolet DNA damage and for DNA apurinic sites, respectively. The levels of both enzyme activities in livers of normal rats were compared to levels in livers of rats fed N-2-acetylaminofluorene. At critical stages of the carcinogenic regimen levels of both endonuclease activities were normal. This, together with other data, suggests that depression of excision-repair of DNA damage does not take place during experimental carcinogenesis.

Nephrogenic metaplasia of the ureter.

We present what, to our knowledge, is the first reported case of nephrogenic metaplasia of the ureter. Nephrogenic metaplasia involves the transitional epithelium of the urinary tract and results in the formation of epithelial tubules, which are histologically similar to renal tubules. Special stains demonstrated both intracellular and intraluminal mucin. Ultrastructurally, the lesion consisted of epithelial cells with sparse microvilli and a thick basal lamina. The criteria for diagnosis and differentiation from carcinoma are discussed.

Modifications of circular DNA by photoalkylation.

The effects of photoalkylation on superhelical PM2 DNA were examined. The chief product was 8-(2-hydroxy-2-propyl)guanine, formed exclusively in sequences of alternating purines and pyrimidines. Other purine damages included 8-(2-hydroxy-2-propyl)adenine and smaller quantities of two uncharacterized adenine products. DNA strand breaks were formed with increasing irradiation. A small quantity of thymine-containing photodimers was formed. Photoalkylation of poly(dG-dC):poly(dG-dC) reduced the concentration of salt required to effect inversion of the circular dichroic spectrum. This suggests that photoalkylation induces the transition of poly(dG-dC):poly(dG-dC) from the right-handed B form of DNA to the left-handed Z form.

Formation of purine photoproducts in a defined human DNA sequence.

The formation of DNA base damages by broad spectrum ultraviolet irradiation (250-400 nm) was investigated using a defined sequence of human DNA. The irradiated, 92 base pair, 3'-end of the human alphoid segment was incubated with an enzyme fraction purified from bacteriophage T4-infected E. coli. As previously reported, analysis of reaction products by sequencing gels showed enzymic incision of purine-containing photoproducts as well as pyrimidine cyclobutane photodimers. The purine-incising activity does not require metal ions and was unaffected by beta-mercaptoethanol or dithiothreitol. The formation of the purine photoproducts is independent of buffer; these lesions are produced by irradiation of DNA in Tris, Hepes or phosphate buffers. They are produced at biologically significant wavelengths between 260 to 300 nm. Only low levels were detected above or below this range. The formation of purine photoproducts is dose dependent with similar yields at some specific loci to pyrimidine dimers. These results suggest that purine-containing photoproducts could be of consequence in ultraviolet carcinogenesis.

Wavelength dependence of DNA incision by a human ultraviolet endonuclease.

The wavelength dependence of an ultraviolet irradiation of the DNA substrate for a human endonuclease was determined. Sites of DNA incision for all UVB and UVC wavelengths examined were at cytosines which were neither cyclobutane pyrimidine dimers nor 6-4'-(pyrimidin-2-one)pyrimidines. The optimal wavelengths for formation of these cytosine photoproducts were between 270 and 295 nm. This human endonuclease therefore has a similar ultraviolet substrate specificity to endonuclease III.

Synthesis and properties of DNA purine dehydrodimers: 8-8-bideoxyribonucleosides and 8-8-bideoxyribonucleotides.

Purine dimers are formed by oxidation of DNA. There is evidence that these dimers are not repaired by cells from the human disease xeroderma pigmentosum. It has been suggested that unrepaired purine dimers are involved in the etiogenesis of internal cancers and neural degeneration that are observed in this disease. In order to study the properties and biological consequences of such moieties, these compounds were synthesized: 8-8-(2'-deoxyadenosyl)-2'-deoxyadenosine; 8-8-(2'-deoxyadenosyl)-2'-deoxyadenosine-5'-monophosphate; 8-8-(2'-deoxyadenosyl)-2'-deoxyguanosine; 8-8-(2'-deoxyadenosyl)-2'-deoxyguanosine-5'-monophosphate; 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine; 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine-5'-monophosphate; 8-8-(2'-deoxyguanosyl)-2'-deoxyadenosine, and 8-8-(2'-deoxyguanosyl)-2'-deoxyadenosine-5'-monophosphate. Following purification, they were characterized by mass spectrometry and nuclear magnetic resonance studies. Ultraviolet, fluorescence, and circular dichroic spectra of these products were established. The behavior of these photoproducts in various chromatographic systems was elucidated. Syntheses of purine dimers and descriptions of their properties can aid the studies of their possible formation in, and excision from, oxidized DNA.

Rates of heat-induced pyrimidine alterations in synthetic polydeoxyribonucleotides.

Hydrolytic damages to DNA can occur at physiological conditions. The possible role of DNA conformation on the distribution of such alterations of pyrimidines was investigated. Model compounds used were the synthetic alternating copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC). Base damages were assayed by paper chromatography using polymers radioactively labeled in cytosine. Conformational changes were assayed by circular dichroic spectral changes. Incubation and heating of the polymers in 1 mM MnCl2 caused the spectral shift reported for the left-handed Z-DNA conformation in the alternating copolymer and the change reported for the triple helix in the homopolymer. After incubation in 85 degrees C, incidences of base damages were compared between the polymers. The presence of manganese reduced depyrimidination in both polymers. Rates of cytosine deamination to uracil were substantial and did not vary among the various conformational states.

Rates of heat-induced DNA purine alterations in synthetic polydeoxyribonucleotides.

Damage to DNA by heat can occur at physiological conditions. The effects of the varying conformational states adopted by double-stranded DNA on the incidences and distributions of thermally induced hydrolytic purine alterations are unknown. The possible role of conformational changes on damage by heat to purines in DNA polymers was therefore investigated. Model compounds used were the synthetic alternating copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC). Base damages were assayed by high performance liquid chromatography using polymers radioactively labeled in guanine. Conformational states were assayed by circular dichroic spectral changes. Incubation and heating of the polymers in 1 mM Mn2+ caused the spectral shift reported for the left-handed Z-DNA conformation in the alternating copolymer and the change reported for the triple helix in the homopolymer. After incubation at 85 degrees C., incidences of base damages were compared between the polymers. No deamination of guanine to xanthine was observed under any conditions. The presence of manganese reduced depurination in both polymers. Rates of guanine imidazole ring openings to yield 2,6-diamino-4-hydroxy-5-formamidopyrimidine were increased in the presence of the cation and constituted the chief form of purine damage in the homopolymer. Therefore, the distribution of heat-induced DNA alterations within the genome may be determined by DNA conformational states. This observed opening of purine imidazole rings in the presence of manganese ions may have mutagenic consequences and may be involved in carcinogenesis by metals.

Differential cell cycle modulation of human DNA glycosylases against oxidized pyrimidines.

Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair of oxidized bases in human cells is initiated by DNA glycosylases which remove them from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme, termed a redoxyendonuclease, has glycosylase activity against many modified DNA pyrimidines. The regulation of these enzymes in proliferating human cells was examined. Both glycosylases were assayed in serum-stimulated WI-38 cells by measurements of direct release of modified free bases from their respective DNA substrates. There was no significant variation of 5-hydroxymethyluracil-DNA glycosylase activity during the cell cycle. However, the glycosylic activity of the redoxyendonuclease was stimulated with DNA synthesis. This activity again increased at the beginning of a second cell cycle. Therefore, the glycosylases that initiate excision repair of oxidized DNA are subject to different controls during the cell cycle.

Glycosylases that excise modified DNA pyrimidines in young and senescent human WI-38 fibroblasts.

Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair in human cells is initiated by DNA glycosylases which remove oxidized bases from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme has glycosylic activity against many ring-saturated DNA pyrimidines. Levels of these enzymes were examined in WI-38 fibroblasts of different culture ages. All glycosylases were assayed by measurements of direct release of modified free bases from their respective DNA substrates. Levels of 5-hydroxymethyluracil-DNA glycosylase were reduced in aging cells. Specific activities of the glycosylase that releases ring-saturated pyrimidines and of uracil-DNA glycosylase were not substantially altered in senescent cells. Therefore, although aging cells might have reduced excision of DNA 5-hydroxymethyluracil, there is no overall age-dependent decrease of DNA glycosylase activities.

Excision of ultraviolet-induced photoproducts of 5-methylcytosine from DNA.

Transition mutations at DNA 5-methylcytosines, congregated at CpG islands, are implicated in the etiogenesis of human diseases. Formation of 5-methylcytosine hydrate (5-methyl-6-hydroxy-5,6-dihydrocytosine) by hydration of the 5,6 double bond of 5-methylcytosine has been suggested as an intermediate in a possible mechanism of deamination to thymine. Ultraviolet irradiation of DNA yields pyrimidine hydrates, which are removed by repair glycosylases. We have identified 5-methylcytosine photoproducts following their excision from DNA by E. coli endonuclease III. Poly(dG-[3H]5-medC):poly(dG-[3H]5-medC) was irradiated and reacted with the enzyme. Radiolabeled photoproduct releases were directly proportional to irradiation doses and enzyme concentrations. These were identified as cis-thymine hydrate (6-hydroxy-5,6-dihydrothymine) and trans-thymine hydrate. Recovery of thymine hydrates is consistent with hydration of pyrimidines. Subsequent heating (which converts thymine hydrates to thymines) and chemical sequencing of an irradiated, 3' end-labeled, synthetic DNA strand demonstrated the appearance of thymine at the 5-methylcytosine site. These results demonstrate a mechanism for deamination of DNA 5-methylcytosine via hydration of the 5,6 double bond, putatively yielding 5-methylcytosine hydrate; this deaminates to thymine hydrate, and loss of water yields thymine formation at the 5-methylcytosine site. Identification of these DNA 5-methylcytosine modified moieties indicates a possible molecular mechanism for the frequent transition mutations found at CpG loci.

Inhibition of DNA polymerase activity by thymine hydrates.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We have demonstrated formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in UV-irradiated poly(dA-dT):poly(dA-dT). Both are released from DNA as free bases by bacterial and human glycosylases. Thymine hydrates are stable in DNA and can be detected in control, unirradiated substrates. We examined the effects of thymine hydrates in UV-irradiated substrate poly(dA-dT):poly(dA-dT) on E. coli DNA polymerase I activity. Enzymic incorporation of labeled thymidine-5'-monophosphate significantly decreased with increasing UV dose. Reversal of DNA thymine hydrates to thymines by mild heating of the substrate prior to enzymic reaction resulted in partial recovery of nucleotide incorporation. Cyclobutane thymine dimers are formed between non-adjacent thymines in UV-irradiated poly(dA-dT):poly(dA-dT). These are responsible for the incomplete recovery of DNA polymerase activity following heating due to their heat stability. Analyses of the irradiated and hydrolyzed substrate also demonstrated formation of minor yields of photoproducts formed by covalent linkage of adjacent thymines and adenines by UV-irradiation. Therefore, the thymine hydrates formed in UV-irradiated DNA partially inhibit polymerase activity during DNA synthesis and thus could be potentially lethal if unrepaired.

Reduced 5-hydroxymethyluracil-DNA glycosylase activity in Werner's syndrome cells.

Werner's syndrome (WS) is an autosomal recessive disease marked by early symptoms of accelerated aging. There is evidence indicating accumulation of oxidized DNA bases to be a major factor in cellular aging. The first step of excision repair of such bases in human cells is their removal from DNA by glycosylases. 5-Hydroxymethyluracil (HMU)-DNA glycosylase excises HMU from DNA; another glycosylase removes many non-aromatic pyrimidine derivatives. Levels of glycosylases that excise oxidized pyrimidines from DNA were compared between confluent and proliferating populations of WS cells, age-matched controls, and young control cells. They were assayed by measurements of direct release of free bases from their respective DNA substrates. Specific activities of the glycosylase that releases various modified pyrimidines and of uracil-DNA glycosylase (which removes uracil from DNA) were essentially the same in all cell lines. Cell cycle variations of these enzymes also did not differ between WS and control cells. HMU-DNA glycosylase specific activity was reduced in WS cells. Reduction of HMU-DNA glycosylase has been described in senescent human WI-38 cells. Therefore, while neither WS nor senescent cells have overall deficiencies of DNA glycosylase activities, they both might have reduced excision of HMU from DNA. This indicates a possible role of HMU accumulation in the aging process.

Effects of 5-azacytosine in DNA on enzymic uracil excision.

PBS-2 phage DNA, which contains uracil in place of thymine, was used as substrate for both purified B. subtilis uracil-DNA glycosylase and a crude extract from M. luteus. Addition of [3H]5-azacytidine to the medium after phage infection resulted in substitution of 1.2% azacytosine for cytosine in DNA. Substrate DNA was also labeled with [14C]uracil. Neither enzyme preparation released tritiated bases from DNA. Analysis by S1 nuclease digestion show no increase in single-strandedness of the modified DNA. Enzymic release of uracil by the M. luteus extract was reduced by about 50% from the substituted substrate. By contrast, the rate of uracil excision by the purified enzyme was unaffected by the presence of DNA 5-azacytosine.