The reaction of iron(II) EDTA with hydrogen peroxide produces the hydroxyl radical, which has proven to be a powerful and widely- used chemical probe of the structure of protein-DNA and protein- RNA complexes. The hydroxyl radical also is produced by the interaction of ionizing radiation with water, and as such is the proximate chemical species that mediates radiation damage to DNA. The long-term goal of this project is to make use of the chemistry of the hydroxyl radical, produced either using the iron(II) EDTA/hydrogen peroxide system or by gamma irradiation, to make high-resolution chemical """"""""images"""""""" of complicated functioning protein-DNA complexes. Systems to be studied include actively-transcribing RNA polymerase, a set of copper metalloregulatory factors from yeast, and the Z-DNA-binding, RNA -editing enzyme ADAR1. A collateral goal of the project is to use new knowledge on the structure and properties of DNA damaged by the hydroxyl radical to isolate and characterize cellular proteins that initially recognize radiation damage to the DNA backbone.
The Specific Aims of the project are: (1) to use the hydroxyl radical as a chemical probe to dissect the interactions that the DNA-binding subdomains of the yeast copper metalloregulatory factors Amt1, Ace1, and Mac1 make with DNA; (2) to prepare a set of three specifically-lesioned DNA oligonucleotides and use them to isolate eukaryotic proteins which recognize oxidative damage to the DNA backbone that is induced by ionizing radiation; (3) to use the results of missing nucleoside experiments on transcribing RNA polymerase performed in the past grant period to prepare specifically-gapped template DNA molecules for analysis of the effect of template structure on transcription. As well, DNA templates with bent and straight adenine tracts will be prepared for studies on the effect of global DNA structure on transcription; (4) to perform hydroxyl radical footprinting experiments on the complex of the RNA editing enzyme ADAR1 with left-handed Z-DNA, with the eventual goal of performing chemical probe experiments to define the structure of a functioning RNA editing system. The results of this project will provide new information on how cells recognize damage to the genome cause by ionizing radiation. As well, the studies to be undertaken on RNA polymerase, the yeast copper metalloregulatory factors, and ADAR1 will give new structural insight into some of the complicated protein-DNA """"""""machines"""""""" which the cell uses to decode the information in the genome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041930-13
Application #
6519322
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Lewis, Catherine D
Project Start
1989-04-01
Project End
2004-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
13
Fiscal Year
2002
Total Cost
$256,990
Indirect Cost
Name
Boston University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Boston
State
MA
Country
United States
Zip Code
02215
Wang, Qun; Tullius, Thomas D; Levin, Judith R (2007) Effects of discontinuities in the DNA template on abortive initiation and promoter escape by Escherichia coli RNA polymerase. J Biol Chem 282:26917-27
Danford, Andrew J; Wang, Dong; Wang, Qun et al. (2005) Platinum anticancer drug damage enforces a particular rotational setting of DNA in nucleosomes. Proc Natl Acad Sci U S A 102:12311-6
Frazee, Richard W; Taylor, Jennifer A; Tullius, Thomas D (2002) Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm. J Mol Biol 323:665-83
Levin, J R; Blake, J J; Ganunis, R A et al. (2000) The roles of specific template nucleosides in the formation of stable transcription complexes by Escherichia coli RNA polymerase. J Biol Chem 275:6885-93
Jamison McDaniels, C P; Jensen, L T; Srinivasan, C et al. (1999) The yeast transcription factor Mac1 binds to DNA in a modular fashion. J Biol Chem 274:26962-7
Widlund, H R; Kuduvalli, P N; Bengtsson, M et al. (1999) Nucleosome structural features and intrinsic properties of the TATAAACGCC repeat sequence. J Biol Chem 274:31847-52
Draganescu, A; Tullius, T D (1998) The DNA binding specificity of engrailed homeodomain. J Mol Biol 276:529-36
Draganescu, A; Levin, J R; Tullius, T D (1995) Homeodomain proteins: what governs their ability to recognize specific DNA sequences? J Mol Biol 250:595-608
Kimball, A S; Kimball, M L; Jayaram, M et al. (1995) Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence. Nucleic Acids Res 23:3009-17
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

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