beta-Amyloid protein induces the formation of purine dimers in cellular DNA.

Experimental evidence implicates oxidative free radical reactions as central in the processes of neurodegenerative diseases. In particular, cellular interactions with the beta-amyloid protein have been linked to neuron cell death in Alzheimer's disease. Also, uncharacterized dimeric purine moieties have been detected in oxidized DNAs. It has been suggested that inadequate excision-repair of such products plays a functional role in the neurological degeneration observed in familial Alzheimer's disease, Down's syndrome, and xeroderma pigmentosum. Therefore, in order to obtain a reagent to monitor the presence of such products, the purine dimer 8-8-(2'-deoxyguanosyl)-2'-deoxyguanosine-5'-monophosphate was used as a hapten for elicitation of rabbit anti-purine dimer antiserum. This antiserum specifically recognizes various purified 8-8-bideoxyribonucleosides and 8-8-bideoxyribonucleotides. We found that DNA oxidized by the Fenton reaction is specifically recognized by this antiserum. This reagent can therefore be used to demonstrate formation and excision of DNA purine dimers. Moreover, incubation of cultured rat pheochromocytoma PC-12 cells with the beta-amyloid protein resulted in formation of these purine dimers in cellular DNA. These dimers were subsequently removed from cellular DNA. From these results we conclude that the free radicals generated by A beta cause oxidative DNA alterations including purine dimers. Deficient repair of this type of DNA damage might result in neural cell loss via apoptosis. Our findings suggest mechanisms for the roles of beta-amyloid and oxidative free radicals in neurodegenerative diseases and the role of DNA excision-repair in the prevention of lethal neurotoxicity.

Human DNA-demethylating activity: a glycosylase associated with RNA and PCNA.

We have partially purified and characterized the 5-methylcytosine removing activity (5-meC-DNA Glycosylase) from HeLa cells with 700-fold enrichment. This activity cleaves DNA specifically at fully methylated CpG sites. The mechanism of 5-meC removal is base excision from fully methylated CpG loci on DNA, producing abasic sites. Hemi-methylated DNA is not a substrate. A prominent 52 KDa protein is present in all partially purified fractions. This activity is tightly associated with other nuclear factors and proteins, which resulted in differential fractionation of this activity on ion exchange columns. One nuclear factor associated with this activity is identified as RNA. Another nuclear protein, proliferating cell nuclear antigen (PCNA) is also associated with this enzyme. Glycosylic removal of 5-meC from DNA by this activity could be involved in the regulation of transcription, replication, differentiation, and development through resultant hypomethylation of DNA.

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.

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.

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.

Enzymic removal of 5-methylcytosine from DNA by a human DNA-glycosylase.

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.

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.

Stability of DNA thymine hydrates.

Pyrimidine hydrates are products of ultraviolet irradiation of DNA. We have already demonstrated the formation of both cis-thymine hydrate and trans-thymine hydrate (6-hydroxy-5,6-dihydrothymine) in irradiated poly(dA-dT):poly(dA-dT). These are released from DNA as free bases by bacterial or human glycosylases. Thymine hydrate stabilities were studied in irradiated DNA substrates using purified E. coli endonuclease III as a reagent for their removal. After irradiation, substrate poly(dA-dT):poly(dA-dT), radiolabeled in thymine, was incubated at 50, 60, 70 or 80 degrees C, cooled, and then reacted with the enzyme under standard conditions. Thymine hydrates were assayed by enzymic release of labeled material into the ethanol-soluble fraction. Their identities were confirmed by high performance liquid chromatography. The decay of thymine hydrates in heated DNA followed first-order kinetics with a k = 2.8 x 10(-5)/sec at 80 degrees C. These hydrates were also detected in lesser quantities in the unirradiated, control substrate. Extrapolation from an Arrhenius plot yields an estimated half-life of 33.3 hours at 37 degrees C for DNA thymine hydrates. Such stability, together with their formation in unirradiated DNA, suggest thymine hydrates to be formed under physiological conditions and to be sufficiently stable in DNA to be potentially genotoxic. This necessitates their constant removal from DNA by the excision-repair system.

Ultraviolet-induced thymine hydrates in DNA are excised by bacterial and human DNA glycosylase activities.

Ultraviolet irradiation of DNA results in various pyrimidine modifications. We studied the excision of an ultraviolet thymine photoproduct by Escherichia coli endonuclease III and by a preparation of human WI-38 cells. These enzymes cleave UV-irradiated DNA at apyrimidinic sites formed by glycosylic removal of the photoproduct. Poly(dA-[3H]dT).poly(dA-[3H]dT) was UV irradiated and incubated with purified E. coli endonuclease III. 3H-Containing material was released in a manner consistent with Michaelis-Menten kinetics. This 3H-labeled material was determined to be a mixture of thymine hydrates (6-hydroxy-5,6-dihydrothymine), separable from unmodified thymine by chromatography in three independent systems. Both cis-thymine hydrate and trans-thymine hydrate were chemically and photochemically synthesized. These coeluted with the enzyme-released 3H-containing material. No thymine glycol was released from the UV-irradiated polymer. Similar results were obtained with extracts of WI-38 cells as the enzyme source. The release of thymine hydrates by both glycosylase activities was directly proportional to the amount of enzyme and the irradiation dose to the DNA substrate. These results demonstrate the modified thymine residues recognized and excised by endonuclease III and the human enzyme to be a mixture of cis-thymine hydrate and trans-thymine hydrate. The reparability of these thymine hydrates suggests that they are stable in DNA and therefore potentially genotoxic.

Excision of cytosine hydrates from Z-DNA.

Ultraviolet irradiation of DNA produces cytosine hydrate, released as a free base by E. coli endonuclease III. Cytosine hydrate excision was investigated by assaying photoproduct release from cytosine-radiolabeled, irradiated poly(dG-dC):poly(dG-dC). Conformational shifts between B-DNA and Z-DNA were affected by heating the polymer in either nickel chloride or cobaltous chloride, and were determined by circular dichroism. Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations. Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation. Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by these DNA conformational states.

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.

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.

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.

Cytosine photoproduct-DNA glycosylase in Escherichia coli and cultured human cells.

Ultraviolet irradiation of DNA produces a variety of pyrimidine base damages. The activities of Escherichia coli endonuclease III and a human lymphoblast endonuclease that incises ultraviolet-irradiated DNA at modified cytosine moieties were compared. Both the bacterial and human enzymes release this cytosine photoproduct as a free base. These glycosylase activities are linear with times of reaction, quantities of enzyme, and irradiation dosages of the substrates. Both enzyme activities are similarly inhibited by the addition of monovalent and divalent cations. Analysis by DNA sequencing identified loci of endonucleolytic incision as cytosines. These are neither cyclobutane pyrimidine dimers, 6-(1,2-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4(1H,3H)-pyrimidinediones, nor apyrimidinic sites. This cytosine photoproduct is separable from unmodified cytosine by high-performance liquid chromatography. This separation should facilitate identification of this modified cytosine and elucidation of its biological significance.

A human endonuclease incises ultraviolet-irradiated DNA at cytosines and oxidized DNA at thymines.

Both ultraviolet irradiation and oxidation of DNA produce a variety of pyrimidine base damages. A human endonuclease recognizes such altered bases on these DNA substrates. This human endonuclease incises ultraviolet-irradiated DNA exclusively at sites of photochemically modified cytosines. The precise sites of incision by the human enzyme were determined by DNA sequencing. Chemically oxidized DNA was incised exclusively at thymine loci. The degree of enzymic cleavage at cytosine photoproducts was identical at each site. However, the extent of incision at selected oxidized thymine residues varied within the DNA sequence. These results indicate that the distribution of thymine oxidative modifications is influenced by the neighboring DNA bases.

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.