Dna Damage, Dna Repair And Alcohol

Alcohol Abuse And Alcoholism

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Abstract

Cyclopurines as Candidate Neurodegenerative DNA Lesions in Xeroderma Pigmentosum Studies in the past year have continued to make progress in understanding the biological significance of a novel class of oxidative DNA lesions called cyclopurines (cPu) that are formed in DNA as a result of the hydroxyl radical. These lesions are unique amongst oxidative DNA lesions in that they are specifically repaired by the nucleotide excision repair pathway. We have previously proposed that the accumulation of cyclopurine lesions on the transcribed strand of active genes is responsible for the neurodegeneration observed in patients with xeroderma pigmentosum who lack the capacity to carry out NER. In our previous work, we showed that a single cyclopurine can prevent binding of the TATA binding protein to a TATA box in vitro, and also strongly reduce gene expression in vivo. Katie Krone in our laboratory has now extended this work showing that a single cyclopurine lesion can completely block the binding of CREB, heat-shock factor, and NF-kappa B to target sequences, and can partially block the binding of the structure-specific factor HMGI-Y. In addition to effects of cyclopurines on transcription factor binding, we have also investigated the possibility that RNA polymerase II can bypass cyclopurines in living mammalian cells. For this purpose, we created plasmid DNA constructs in which a single lesion was placed on the transcribed strand of a reporter gene, downstream from an intron. The lesion was placed in a unique restriction site to facilitate analysis of mutants. The lesion containing construct, or a lesion-free control, were transfected into NER -deficient cells, and isolated RNA analyzed by RT-PCR followed by restriction digestion and sequencing. Using this approach, we have obtained evidence that some abnormal (mutant) RNA transcripts can be produced when the polymerase bypasses the cyclopurine lesion in vivo. These results represent the first evidence that DNA lesions of the type that are repaired by the NER pathway can stimulate transcriptional mutagenesis. We are now writing a manuscript describing these results, and using the same methodology to study the bypass of other DNA lesions that are repaired by RNA pol II in vivo. In addition, we are currently buttressing this in vivo results in in vitro systems. A collaborative study with the laboratory of Dr. Kiyoji Tanaka has shown that a single cyclopurine can completely block transcription by purified RNA polymrease II using dC-tailed constructs. In additon, Anoop Patel in the lab has obtained evidence indicating that the cyclopurine lesion blocks transcription by RNA polymrease II in nuclear extracts. Using both systems, we are working to characterize the nucleotides that the polymrease incorporates opposite the lesion in vitro, and compare these with the in vivo results with the lesion in the same sequence location. In addition to focusing on the effect of the lesion when encountered by the RNA polyrmease in the elongation mode, we have also prepared additional constructs in which the lesion is located in a promoter proximal position 18 nucleotides downstream from the transcription start site. Using these constructs, we have found that while the lesion resutls in the same decrease in reporter gene expression in either location when transfected into cells from and XPA patient, the effect of the lesion is less severe when located at position +18 in cells from an XPD patient. This observation suggests that, in contrast to XPA cells, XPD cells retain some capacity to repair lesions in promoter-proximal sequences. The molecular basis for this observation in under investigation. Molecular Mechanisms of Neurodegeneration in Other DNA Repair Syndromes Mutations in the genes encoding several proteins involved in the detection of DNA damage, including Mre11 and ATM, lead to neurological disease. Work by other groups has shown that these proteins function as part of the DNA damage responsee in cells. Nearly all of this work has been focused on the DNA damage response in relation to cell-cycle checkpoints. Much less is known about how defects in these proteins lead to neurodegenerative disease. In the past year, we found that the Mre11/Rad50/Nbs1 (MRN) complex proteins are concentrated in large neurons in the juvenile human brain as well as in Bergman glial cells. These observation are consistent with the hypothesis that that the MRN complex plays an ongoing role in postmitotic neurons of the human brain, and that it is the loss of this fucntion which results in neurodegeneration. This manuscript has been submitted. Another neurologic disease beleived to result from a DNA repair defect is Ataxia Telangiectasia (AT). However, there is currently controverys about the subcellular localization of the ATM protein in the Purkinje neurons which are affected in AT patients, and whether the neurological features of the disease are the result of the loss of DNA repair or some other fucntion fo the protein. By Western blotting we have shown that exposure of juveniel rats to ionizing radtion indu ed a dose-dependent activation of ATM in the cerebellum, indicating that the ATM protein in the jevenile rat brain does respond to DNA damage. Onbgoing studies are focused on the cellular and subcellualr sites of ATM activation in the cerebellum. Molecular Mechanisms of Carcinogenesis Resulting From Alcohol Abuse While N2EtdG appears useful as a marker for alcohol-related DNA damage, the biological effects of this lesion are not clear. We have therefore focused our attention on another acetaldehyde related DNA lesion, 1, N2-propanodeoxyguanosine (PdG). This lesion is repaired by NER, and has been shown to cause mutagenesis in mammalian cells. The formation of this lesion from acetaldehyde and deoxyguanosine has been reported to be stimulated by the basic amino acids lysine and arginine as are found in histones. We have found that PdG formation can also be stimulated by the polyamines spermine and spermidine, using concentrations of acetaldehyde as low as 100 micromoral. Polyamine synthesis is strongly correlated with cell division, and is elevated in cells undergoing active DNA synthesis. We therefore speculate that a high abundance of polyamines in rapidly dividing cells favors the formation of the mutagenic PdG adduct from AA, which may be relevant to the mechanisms of alcohol related gastrointestinal cancer. This work was published in Nucleic Acids Research, and was the subject of a news release by the NIH and NIAAA (http://www.nih.gov/news/pr/aug2005/niaaa-03.htm.) Ongoing studies are focused on confirming the polyamine mechanism in living cells.