Dna Damage, Dna Repair And Alcohol

Alcohol Abuse And Alcoholism

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Abstract

Biology of Cyclopurines Studies in the past year continued have made 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. In our previous publication using the 32P-postlabelling assay, we concluded that cyclopurines are present in mammalian tissue at levels on the order of 1 lesion / 10 to the 8 normal nucleotides. Using LC and GC mass spectrometry, Dr. Miral Dizdaroglu and colleagues at NIST obtained considerably higher values, on the order of 1 / 10 to the 7. In a collaborative effort I initiated with Dr. Dizdaroglu to resolve these differences, we have determined that the postlabelling method significantly underestimated cPu levels due to loss of cPu during the procedure. Moreover, we found that the MS assays may also have slightly underestimated the levels of cPu in samples. Thus basal levels of cPu in tissues and mammalian cell in culture are on the order of 1 / 10 to the 7 or higher, further underscoring the biological importance of this unique class of lesions not only in patients with XP, but also in normal individuals. In addition, we are now investigating levels of cPu in the human brain with aging. These studies are presently carried out in collaboration with Dr. Dizdaroglu. However, with the arrival of Jacob Theruvathu in our lab and his development of mass spec assays for endogenous oxidative DNA lesions (see below), future measurements of cPu under different conditions, including after alcohol exposure, will be carried out in our laboratory. We have also obtained 4 monoclonal antibodies against cPu-containing DNA, and are characterizing these antibodies for use in immunocytochemical studies. Lastly, we have entered into collaboration with Dr. Chul-Hee Kang at Washington State University who is currently determining the structure of the cPu lesion using X-ray crystallography. Double-Strand Break Repair and Neurological Disease Patients with the genetic disease ataxia telangiectasia (AT) develop a progressive ataxia during childhood that results from severe loss of Purkinje neurons in the cerebellum. The mutated gene in AT patients, called ATM, encodes a DNA-dependent protein kinase. While all of the known functions of the ATM protein involved interactions with damaged DNA, understanding the mechanisms of neurodegeneration in AT has been complicated by inability of several investigators to detect the ATM protein in the nucleus of the Purkinje neurons that are known to degenerate in this disease. Using biochemical methods, we have now detected ATM in highly purified cerebellar nuclei from human and rodent brain. We have entered into collaboration with Dr. Susan Lees-Miller and the University of Calgary to purify the ATM protein from brain nuclei and examine its catalytic properties. Among the several targets of ATM kinase is a protein called Mre11 that is involved in DSB repair. Interestingly, patients with mutations in Mre11 also develop progressive cerebellar neurodegeneration. Studies begun in the past year have localized Mre11, and related proteins to the nucleus of Purkinje neurons as well as other large neurons in the human brain. These findings have important implications regarding the mechanisms of neurodegeneration in AT and ATLD patients. A manuscript describing this work has been submitted. Understanding the role of the ATM pathway in Purkinje neurons may also have implications for understanding the effects of alcohol in the developing brain. A gene-chip analysis of mRNA expression in an animal model of fetal alcohol syndrome (carried out by Susan Maier and Jim West at Texas A & M University) found that the most highly induced mRNA in the cerebellum of the alcohol treated offspring was Rad9. Rad9 has been shown to be a substrate for phosphorylation by the ATM in response to DNA damage. We have investigated the expression of rad9 in the rodent brain, and observed that it is localized in neuronal nuclei. Previous investigators have found that in cultured cells, rad9 becomes phosphorylated on a specific serine residue, 272, in response to exogenous DNA damage. However, we have found that a fraction of neuronal rad9 is constitutively phosphorylated on serine residue 272. We are proposing that this phosphorylation event represents an endogenous DNA damage response. A manuscript describing these findings is in preparation. A Panel of Assays for Alcohol-Induced DNA Lesions Our working hypothesis in regard to the pathological effects of alcohol in the body is that these effects are mediated, at least in part, by damage to DNA. One specific DNA lesion that has been shown to be present in human alcoholics is N2-ethyl-2?-deoxyguanosine (N2EtdG), which results from the reaction of acetaldehyde with deoxyguanosine. In the past year, Dr. Jacob Theruvathu joined our group to develop a GC-MS assay for N2EtdG, as well as for a panel of other structurally different, oxidative DNA lesions. Dr. Theruvathu has already developed the assay for N2EtdG, in both the base form (N2-ethyl-guanine) and nucleoside, and has synthesized an isotopically labeled form of N2EtdG to act as an internal standard to maximize the precision of the assay. He is continuing to develop assays for several other DNA lesions resulting from oxidative stress(including cyclopurines, see above)and lipid peroxidation. The goal of this work is to develop a panel of assays so that DNA samples from animal models of alcohol exposure, as well as human samples, can be assayed for a variety of DNA lesions simultaneously.