02-Alkyl(methylhethyl) thymidine, 04-alkyl thymidine and-02-alkyl cytidine are formed in vivo by a variety of N-nitroso carcinogens. In all organs and eukaryotic cells examined, these derivatives persist for long periods. The promutagenic nature of each of these pyrimidines has been demonstrated in vitro and in vivo for 04-alkyl T. 04-Methyl and -ethyl thymidines are also directly implicated in initiation of hepato-carcinogenesis. This project is directed toward understanding how the presence of 0-alkyl pyrimidines in DNA or model oligomers affects secondary structure and polymerase recognition in replication. 02- and O4-alkyl thymine will be site-specifically incorporated in template-primer complexes by DNA polymerase of varying fidelity. They will be placed opposite A or G, the known partners, or T, a mismatch. The kinetics of insertion and elongation (km Vmax) will be determined by gel electrophoresis or radiolabeled nucleotide incorporation. Structures of complete duplexes, e.g., a 22-mer, containing 0 to 3 alkyl thymines, will be examined by thermal denaturation, enzyme sensitivity and antibody recognition. A hexamer capable of the B to Z transition will be synthesized with a single alkyl thymine and the result of the substitution studied by circular dichroism. Duplex oligomers with 2-aminopurine opposite 04-alkyl thymine will be used to measure fidelity and structure in the absence of steric hindrance. Fluorescence changes are a sensitive indicator of the degree of stacking and bonding. Sequence effects on kinetics and structure will be determined by varying the template oligomers. Sequences with stability may be less likely to be repaired, so mutation can occur. Preparation of 02-alkyl CDP and CTP will use mild chemical or enzymatic methods to attempt prevention of dealkylation and depyrimidination. If the desired products are obtained, polymers can be made for mispairing studies. Finally, a pair of cell lines, isolated from human brain tumors, will be used to study the time course and extent of 0-alkyl pyrimidine repair. These cells differ greatly in their ability to repair the initial O6-alkyl G adduct formed by chemotherapeutic halonitrosoureas. Either cell one may be suitable for further study of pyrimidine repair mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA042736-08S1
Application #
2090907
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1979-04-01
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1995-03-31
Support Year
8
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Lawrence Berkeley National Laboratory
Department
Anatomy/Cell Biology
Type
Organized Research Units
DUNS #
078576738
City
Berkeley
State
CA
Country
United States
Zip Code
94720
Singer, B (1996) DNA damage: chemistry, repair, and mutagenic potential. Regul Toxicol Pharmacol 23:2-13
Rao, S; Chenna, A; Slupska, M et al. (1996) Replication of O4-methylthymine-containing oligonucleotides: effect of 3' and 5' flanking bases on formation and extension of O4-methylthymine . guanine basepairs. Mutat Res 356:179-85
Pongracz, K; Dosanjh, M K; Singer, B et al. (1994) Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate. Carcinogenesis 15:1371-5
Dosanjh, M K; Chenna, A; Kim, E et al. (1994) All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase. Proc Natl Acad Sci U S A 91:1024-8
Dosanjh, M K; Loechler, E L; Singer, B (1993) Evidence from in vitro replication that O6-methylguanine can adopt multiple conformations. Proc Natl Acad Sci U S A 90:3983-7
Dosanjh, M K; Menichini, P; Eritja, R et al. (1993) Both O4-methylthymine and O4-ethylthymine preferentially form alkyl T.G pairs that do not block in vitro replication in a defined sequence. Carcinogenesis 14:1915-9
Singer, B; Antoccia, A; Basu, A K et al. (1992) Both purified human 1,N6-ethenoadenine-binding protein and purified human 3-methyladenine-DNA glycosylase act on 1,N6-ethenoadenine and 3-methyladenine. Proc Natl Acad Sci U S A 89:9386-90
Dosanjh, M K; Galeros, G; Goodman, M F et al. (1991) Kinetics of extension of O6-methylguanine paired with cytosine or thymine in defined oligonucleotide sequences. Biochemistry 30:11595-9
Singer, B; Essigmann, J M (1991) Site-specific mutagenesis: retrospective and prospective. Carcinogenesis 12:949-55
Dosanjh, M K; Singer, B; Essigmann, J M (1991) Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli. Biochemistry 30:7027-33

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