The photochemistry of oligonucleotides in discrete conformations (I) will be examined to ascertain the secondary structural features required for DNA hyperreactivity with the ubiquitous environmental mutagen and carcinogen, sunlight. Disordered duplex structures proposed for in vitro analysis ([1] a bulge loop, [2] an interior loop and [3] a hairpin known to exist with B and Z helices) form in cells only transiently and at low concentrations but nevertheless may represent regions of DNA with the greatest predilection for genetic transformation. The reactivity of these DNA conformations will be characterized under model conditions that independently and sequentially mimic specific photoprocesses known to occur in vivo. Such comparative studies will help to evaluate the physiological importance of 1) direct nucleotide excitation, 2) DNA photosensitization and 3) photochemical oxidation. This final, oxidative mechanism will be further characterized by studying 4) DNA degradation effected by individual reactive oxygen species including, but not limited to, singlet oxygen generated by photochemical and nonphotochemical methods. Concurrently, an oligonucleotide-directed photosensitizer will be studied for potential use in site specific DNA modification (II). This covalent oligonucleotide-photosensitizer complex should hybridize exclusively to DNA containing a complementary sequence and then catalyze derivatization of only bases adjacent to the bound photosensitizer. Applications of this technique include 1) the preparation of ample quantities of DNA containing single, defined premutagenic lesions for future studies on DNA repair and 2) the cleavage of single-stranded DNA in a highly specific, restriction enzyme-like manner useful in gene manipulation and diagnosis. The DNA sequences used in this proposal are found in the plasmid, pBR322, and will, therefore, facilitate a long range extension of this research towards detailed analysis of the chemical events that potentiate both the formation and repair of premutagenic lesions in vivo. The research will provide important mechanistic information on the origins of medical disorders associated with sunlight exposure.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA043593-01
Application #
3185824
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1986-12-01
Project End
1989-11-30
Budget Start
1986-12-01
Budget End
1987-11-30
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Rokita, S E; Romero-Fredes, L (1992) The ensemble reactions of hydroxyl radical exhibit no specificity for primary or secondary structure of DNA. Nucleic Acids Res 20:3069-72
Rokita, S E; Lau, B; Romero-Fredes, L (1990) Structural dependence of oligonucleotide photooxidation. Biopolymers 29:69-77
Rokita, S E; Romero-Fredes, L (1989) Facile interconversion of duplex structures formed by copolymers of d(CG). Biochemistry 28:9674-9