Walter A. Deutsch

A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities

Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis. A major product of free radical DNA damage is the formation of 8‐oxoguanine, which is a highly mutagenic base modification produced by oxidative stress. Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8‐oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction. The significance of this DNA repair activity acting on 8‐oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8‐oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8‐oxoguanine‐mediated G–>T transversions. The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites. These results raise the possibility that DNA repair may be associated with protein translation.

Differential expression of the apurinic/apyrimidinic endonuclease (APE/ref-1) multifunctional DNA base excision repair gene during fetal development and in adult rat brain and testis

The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease is responsible for the repair of AP sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA binding capability of Jun-Jun homodimers and Fos-Jun heterodimers by altering their redox state and to be involved in calcium mediated transcriptional repression of the parathyroid hormone gene. Previous studies examining the tissue specific distribution of the AP endonuclease (APE) transcript and protein by Northern analysis and enzymatic assays, respectively, have shown that this gene is expressed in all tissues at relatively similar levels. In the current study, adult and fetal rat tissue sections were examined for the expression of the APE transcript in specific subpopulations of cells and during development by in situ hybridization. In the adult brain, the APE transcript showed a widespread, but heterogeneous pattern of expression. Predominant levels of transcript were detected in the suprachiasmatic nuclei, the supraoptic and paraventricular nuclei, the hippocampus and the cerebellum. During fetal development, transcript was detected in all somatic sites examined with very high levels in the thymus, liver and developing brain. Examination of the adult testis indicated that the expression of the transcript varies with the stage of spermatogenesis with the highest levels being present over round spermatids. These results provide evidence that the APE gene is not homogeneously expressed, but rather is found in subpopulations of cells in the brain and testes and during development.

Cigarette tar causes single-strand breaks in DNA

The results of this study demonstrate, for the first time, that cigarette tar causes DNA damage. Incubation in vitro of phage PM2 DNA with aqueous extracts of cigarette tar results in the introduction of DNA single-strand breaks. The effects of protective enzymes and radical scavengers indicate the involvement of active oxygen species. Although the semiquinone components of tar reduce dioxygen forming superoxide radicals and hydrogen peroxide, our results suggest that hydroxyl radicals formed via metal catalyzed decomposition of hydrogen peroxide are ultimately responsible for the DNA lesions. Our results also suggest that the metals in tar are reduced by the semiquinone components of tar and by superoxide at comparable rates.

The Drosophila ribosomal protein S3 contains a DNA deoxyribophosphodiesterase (dRpase) activity.

The Drosophila ribosomal protein S3 has been previously demonstrated to cleave DNA containing 8-oxoguanine residues and has also been found to contain an associated apurinic/apyrimidinic (AP) lyase activity that cleaves phosphodiester bonds via a β, δ-elimination reaction. The activity of this protein on DNA substrates containing incised AP sites was examined. A glutathione S-transferase fusion protein of S3 was found to efficiently remove sugar-phosphate residues from DNA substrates containing 5′-incised AP sites as well as from DNA substrates containing 3′-incised sites. Removal of 2-deoxyribose-5-phosphate as 4-hydroxy-2-pentenal-5-phosphate from a substrate containing 5′-incised AP sites occurred via a β-elimination reaction, as indicated by reaction of the released sugar-phosphate products with sodium thioglycolate. The reaction for the removal of 4-hydroxy-2-pentenal-5-phosphate from the substrate containing 3′-incised AP sites was dependent on the presence of the Mg2+ cation. These findings suggest that the S3 ribosomal protein may function in several steps of the DNA base excision repair pathway in eukaryotes and may represent an important DNA repair function for the repair of oxidative and ionizing radiation-induced DNA damage.

Complexes of Pd(II), Pt(II), Cu(II), Co(II) and Zn(II) chlorides with 6, 6′-dimethyl-2, 2′-dipyridyl

Abstract The synthesis of complexes of the general form M(dmdp)Cl2, where dmdp is 6, 6′-dimethyl-2, 2′-dipyridyl and M is Pd(II), Pt(II), Cu(II), Co(II) and Zn(II) from non-aqueous solvents is reported. Also presented is a new convenient synthesis of dmdp. The characteristics of these complexes to produce nicks in the phosphodiester backbone of DNA are evaluated.

The role of combustion-generated radicals in the toxicity of PM2.5

Electron paramagnetic resonance analyses of samples of ambient, fine particulate matter (PM 2.5 ), as well as fly-ashes and soots from a wood-burning fireplace, a boiler cofired with chlorinated organics, a pilot-scale hazardous waste incinerator, and laboratory combustion of various halocarbons reveal that they contain stable free radicals. All of the radical systems were indefinitely stable in air. Extracts of PM 2.5 and the incinerator ash caused damage to DNA, both in vitro and in cell systems. Cellular damage to DNA was prevented by addition of the enzymes superoxide dismutase and/or catalase that destroy the superoxide radical and hydrogen peroxide, respectively. This type of DNA damage and protection is observed for the semiquinone radicals in cigarette tar. Therefore, we have tentatively assigned the structure of the observed radicals as semiquinones. In solution, semiquinone radicals undergo redox cycling, reducing oxygen and forming superoxide radical and hydrogen peroxide, and ultimately the hydroxyl radical that can initiate DNA damage. Since ambient PM 2.5 is primarily composed of combustion-generated particulate matter, our results imply that combustion sources produce semiquinone-type radicals that persist on fine particles in the atmosphere. The fine particles provide a carrier for deposit of the radicals deep in the human respiratory tract. These radicals then initiate immune system responses that can trigger the production of further radicals as well as other species that can damage DNA or induce damage to the respiratory tract.

Repair of alkylated DNA: Drosophila have DNA methyltransferases but not DNA glycosylases

SummaryDNA methyltransferase activity has been identified in crude extracts of Drosophila melanogaster pupae for the removal of methyl groups from O-6 methylguanine appearing in alkylated DNA. Additionally, N-7 methylguanine and 3 methyladenine appear to the uniquely susceptible to methyltransferase activity that resides in Drosophila pupae. Consistent with this, tests to detect DNA glycosylase activity for the repair of the latter two modified bases was unsuccessful, even though a substantial loss of methyl groups from these bases was observed. Conversely, the repair of methylated purines was not detected in extracts of Drosophila embryos. The removal of methyl groups from methylated purines was dependent upon incubation temperature and was proportional to the amount of protein added to reaction mixtures. Results indicate that the methyl group is attached to protein during the repair of methylated DNA, suggesting that it is similar to the O6-methylguanine-DNA methyltransferase identified in other organisms. Although other explanations are possible, the inability to detect DNA glycosylase activity suggests that Drosophila may not rely on base excision repair for the removal of modified or nonconventional basis in DNA.

Apurinic DNA endonucleases from Drosophila melanogaster embryos.

SummaryApurinic DNA endonuclease activity from Drosophila melanogaster embryos was resolved into two separable forms by phosphocellulose chromatography, one which flowed through the column (Fraction I) and the other which was retained and eluted at approximately 200 mM potassium phosphate (Fraction II). Both fractions, purified further by glycerol gradient sedimentation, were found to introduce nicks into DNA that were specific for and equal in number to the alkali-labile sites in depurinated DNA. They had similar apparent Km values for apurinic sites (0.7 nM apurinic sites for Fraction I and 0.8 nM for Fraction II), but differed with respect to optimal pH, Mg++ requirement and sensitivity to EDTA.

An invitro characterization of interstrand cross-links in DNA exposed to the antitumor drug cis-dichlorodiammineplatinum(II)

Abstract The cis -isomer of the antitumor drug dichlorodiammineplatinum(II) [ cis -Pt(II)] was tested for its abilty to introduce nicks (single-strand breaks) into supercoiled PM2 DNA. Whereas incubations up to 24 h show no indication of cis -Pt(II)-treated DNA having single-strand breaks, DNA interstrand cross-links were detected in the first 15 min of incubation. Furthermore, the formation of DNA interstrand cross-links was both inhibited and fully reversed after incubation with 2 mM thiourea.

Genomic Structure and Characterization of the Drosophila S3 Ribosomal/DNA Repair Gene and Mutant Alleles

The Drosophila S3 protein is known to be associated with ribosomes, where it is thought to play a role in the initiation of protein translation. The S3 protein also contains a DNA repair activity, efficiently processing 8-oxoguanine residues in DNA via an N-glycosylase/apurinic-apyrimidinic (AP) lyase activity. The gene that encodes S3 has previously been localized to one of the Minute loci on chromosome 3 in Drosophila. This study focused on the genomic organization of S3 at M(3)95A, initial promoter characterization, and analysis of three mutant alleles at this locus. The S3 gene was found to be a single-copy gene 2 to 3 kb in length and containing a single intron. The upstream 1.6-kb region was analyzed for promoter activity, identifying a presumptive regulatory domain containing potential enhancer and suppressor elements. This finding is of interest, as the S3 gene is constitutively expressed throughout development and mRNA is most likely maternally inherited. Lastly, three Minute alleles from the same locus were sequenced and two alleles found to contain a 22-bp deletion in exon 2, resulting in a truncated S3 protein, although wildtype levels of S3 mRNA and protein were detected in the viable heterozygous Minute alleles, possibly reflecting dosage compensation.