The mechanisms governing the generation and distribution of metal-catalyzed OH. in the immediate vicinity of chromatin remain unclear due in part to lack of methods sufficiently specific to study these short-lived radicals. These researchers have developed an optical methodology to address this problem. Novel fluorescent probes for the detection of OH. have been developed by these researchers and these probes will be incorporated into specific sites of chromatin and genes of high metal affinity within chromatin to allow the simultaneous and independent detection of (i) metal catalyzed OH. formation; and (ii) chromatin conformation. Furthermore, chromatin-damage assays can be subsequently carried out on the same sample in order to relate cause (OH.) and effect (damage). This method will be used in isolated chromatin to (i) quantitate the accessibility of OH. catalyzed by copper, iron or chromium to specific chromatin regions, and (ii) correlate the optical information obtained in particular chromatin conformations with chromatin damage scored on the same samples in order to evaluate the distribution and significance of metal-catalyzed OH.. In a precisely characterized minichromosome system, the microdistribution of metal-catalyzed OH. within individual histones, DNA and the 5S rRNA gene and the binding site of the transcription factor TFIIIA on this gene will be examined. The project will also examine the feasibility of developing the method for use in living cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA072046-05
Application #
2895705
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Okano, Paul
Project Start
1996-08-20
Project End
2001-05-31
Budget Start
1999-06-01
Budget End
2001-05-31
Support Year
5
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
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Chakrabarti, S; Mahmood, A; Makrigiorgos, G M (1999) Fluorescent labelling of closely-spaced aldehydes induced in DNA by bleomycin-Fe(III). Int J Radiat Biol 75:1055-65
Chakrabarti, S; Kassis, A I; Slayter, H S et al. (1998) Continuous detection of radiation or metal generated hydroxyl radicals within core chromatin particles. Int J Radiat Biol 73:53-63
Makrigiorgos, G M; Chakrabarti, S; Mahmood, A (1998) Fluorescent labelling of abasic sites: a novel methodology to detect closely-spaced damage sites in DNA. Int J Radiat Biol 74:99-109
Botchway, S W; Chakrabarti, S; Makrigiorgos, G M (1998) Novel visible and ultraviolet light photogeneration of hydroxyl radicals by 2-methyl-4-nitro-quinoline-N-oxide (MNO) and 4,4'-dinitro-(2,2')bipyridinyl-N,N'-dioxide (DBD). Photochem Photobiol 67:635-40
Chakrabarti, S; Mahmood, A; Kassis, A I et al. (1996) Generation of hydroxyl radicals by nucleohistone-bound metal-adriamycin complexes. Free Radic Res 25:207-20