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Yields of damage to C4′ deoxyribose and to pyrimidines in pUC18 by the direct effect of ionizing radiation

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      Abstract

      Our mechanistic understanding of damage formation in DNA by the direct effect relies heavily on what is known of free radical intermediates studied by EPR spectroscopy. Bridging this information to stable product formation requires methods with comparable sensitivities, a criterion met by the 32P-post-labeling assay developed by Weinfeld and Soderlind, [Weinfeld,M. and Soderlind,K.-J.M. (1991) 32P-Postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry, 30, 1091–1097] which when applied to the indirect effect, detected phosphoglycolate (pg) and thymine glycol (Tg). Here we applied this assay to the direct effect, measuring product yields in pUC18 films with hydration levels (Γ) of 2.5, 16 or 23 waters per nucleotide and X-irradiated at either 4 K or room temperature (RT). The yields of pg [G(pg)] for Γ  ∼  2.5 were 2.8 ± 0.2 nmol/J (RT) and 0.2 ± 0.3 nmol/J (4 K), which is evidence that the C4′ radical contributes little to the total deoxyribose damage via the direct effect. The yield of detectable base damage [G(B*)] at Γ  ∼  2.5 was found to be 30.2  ±  1.0 nmol/J (RT) and 12.9  ±  0.7 nmol/J (4 K). While the base damage called B*, could be due to either oxidation or reduction, we argue that two reduction products, 5,6-dihydrouracil and 5,6-dihydrothymine, are the most likely candidates.

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      Oxidative Strand Scission of Nucleic Acids: Routes Initiated by Hydrogen Abstraction from the Sugar Moiety.

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        DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine.

        Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer. Estimates commonly rely on measurements of 8-oxo-2'-deoxyguanosine (oxo8dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair. Here we examine difficulties inherent in the analysis of oxo8dG, identify sources of artifacts, and provide solutions to some of the common methodological problems. A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo8dG. We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo8dG when compared, under equivalent conditions, with a successful nonphenol method. A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats. Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo8dG values. In further studies we show that human urinary 8-oxo-guanine (oxo8Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo8Gua is not derived enzymatically from xanthine oxidase. Lastly, we discuss remaining uncertainties inherent both in steady-state oxo8dG measurements and in estimates of endogenous oxidation ("hit rates") based on urinary excretion of oxo8dG and oxo8Gua.
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          DNA conformation is determined by economics in the hydration of phosphate groups.

          Mixed sequence DNA can exist in two right-handed and one left-handed double helical conformations--A, B and Z. Under conditions of high water activity the B conformation prevails. If the water activity is reduced on addition of salt or organic solvents, transformation occurs to A-DNA or, in DNAs with alternating purine-pyrimidine sequences, to the left-handed Z-DNA. In crystal structure analyses of oligonucleotides, the free oxygen atoms of adjacent phosphate groups along the polynucleotide chain in B-DNA are found at least 6.6 A apart and individually hydrated whereas they are as close as 5.3 A in A-DNA and 4.4 A in Z-DNA, and bridged by water molecules. We suggest that this more economical hydration in A- and Z-DNA compared with B-DNA is the underlying cause of B----A and B----Z transitions.
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            Author and article information

            Affiliations
            1Department of Biochemistry and Biophysics, University of Rochester, Medical Center, Rochester, NY 14642, USA, 2Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, T6G 1Z2, Canada and 3Department of Radiology, University of California at San Diego, La Jolla, CA 92093, USA
            Author notes
            *To whom correspondence should be addressed. Tel: +1 585 275 3730; Fax: +1 585 275 6007; Email: William_Bernhard@ 123456urmc.rochester.edu
            Journal
            Nucleic Acids Res
            Nucleic Acids Res
            nar
            nar
            Nucleic Acids Research
            Oxford University Press
            0305-1048
            1362-4962
            July 2012
            July 2012
            28 March 2012
            28 March 2012
            : 40
            : 13
            : 6060-6069
            3401456
            22467205
            10.1093/nar/gks271
            gks271
            © The Author(s) 2012. Published by Oxford University Press.

            This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

            Counts
            Pages: 10
            Categories
            Genome Integrity, Repair and Replication

            Genetics

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