Transition metal complexes will be used to probe conformational variation along moderately large DNA molecules, several hundred base pairs in length. Two specific systems will be investigated: 1. The antitumor drug cis-diamminedichloroplatinum(II) (Cisplatin) has been shown to bind to sequences of adjacent guanine bases in a restriction fragment of DNA; this binding is sensitive to variations in DNA conformation, such that the sequence d(G6-C-G2) was found not to be a favorable site for platinum binding except the presence of the intercalator ethidium bromide, a molecule that modifies DNA structure. This sensitivity of Cisplatin to DNA conformation will be exploited in two ways. The effect on DNA conformation of the modified nucleic acid bases inosine and 5-methylcytosine will be assessed by substituting these bases for G or C in the d(G6-C-G2) sequence and measuring platinum binding in the presence and absence of ethidium. A change in DNA conformation caused by incorporation of the modified bases should lead to increased platinum binding in the absence of ethidium. Cisplatin binding also will be used as a sensitive reporter of the width of the major groove of DNA in sequences upstream of eukaryotic genes that show evidence of being in unusual conformations by virtue of their extreme nuclease sensitivity. Sequences upstream of the chicken Beta-globin gene, the histone H-5 gene and the chicken pro-Alpha2(1) gene will be studied. 2. The second experimental system to be studied will be a newly-developed method for mapping variations of helical twist along DNA molecules of particular sequence. This technique makes use of the lack of sequence discrimination shown by the hydroxyl radical in its degradation of the backbone of DNA, to allow fragmentation of a DNA restriction fragment bound to a crystalline inorganic surface. A smooth modulation pattern of fragmentation results from the periodic accessibility to the hydroxyl radical of the DNA strands bound to the surface. This pattern may be visualized by separating the products of the fragmentation reaction on a DNA sequencing gel, and analyzed by densitometry. Hydroxyl radical will be produced by a reagent consisting of ferrous EDTA, hydrogen peroxide, and ascorbate. This method will be used to map the helical twist of the eukaryotic upstream sequences mentioned above. These two experiments will offer a way of mapping conformation along any DNA molecule, in order to relate structure to biological activity for DNA.

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National Institute of General Medical Sciences (NIGMS)
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Metallobiochemistry Study Section (BMT)
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Johns Hopkins University
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Guo, Hong; Tullius, Thomas D (2003) Gapped DNA is anisotropically bent. Proc Natl Acad Sci U S A 100:3743-7
Chen, Bingzi; Jamieson, Elizabeth R; Tullius, Thomas D (2002) A general synthesis of specifically deuterated nucleotides for studies of DNA and RNA. Bioorg Med Chem Lett 12:3093-6
Balasubramanian, B; Pogozelski, W K; Tullius, T D (1998) DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc Natl Acad Sci U S A 95:9738-43
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Ganunis, R M; Guo, H; Tullius, T D (1996) Effect of the crystallizing agent 2-methyl-2,4-pentanediol on the structure of adenine tract DNA in solution. Biochemistry 35:13729-32
Price, M A; Tullius, T D (1993) How the structure of an adenine tract depends on sequence context: a new model for the structure of TnAn DNA sequences. Biochemistry 32:127-36
Kimball, A S; Lee, J; Jayaram, M et al. (1993) Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase. Biochemistry 32:4698-701
Price, M A; Tullius, T D (1992) Using hydroxyl radical to probe DNA structure. Methods Enzymol 212:194-219
Churchill, M E; Hayes, J J; Tullius, T D (1990) Detection of drug binding to DNA by hydroxyl radical footprinting. Relationship of distamycin binding sites to DNA structure and positioned nucleosomes on 5S RNA genes of Xenopus. Biochemistry 29:6043-50
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