Base sequence selectivity in the binding of (+)anti-Benzo(a)pyrene diol epoxide to oligodeoxyribonucleotide duplexes

Jason Jay Schwartz, Purdue University

Abstract

The effect of nucleotide sequence on the binding of (+)anti-Benzo (a) pyrene ((+)anti-BPDE) to the exocyclic amino group of deoxyguanosine (dG) was investigated with duplexes formed from self-complementary oligodeoxyribunucleotides of defined sequence. The oligodeoxyribonucleotides had the same base composition but differed in the primary nucleotide sequence. A $\sp{35}$S-postlabelling assay was used to quantitate the total level of (+)anti-BPDE bound to the duplexes. When (+)anti-BPDE was reacted with the oligodeoxyribonucleotide duplexes, non-random binding was observed. The duplex d(ATACGCGTAT) reacted the most extensively with (+)anti-BPDE: over three-fold more (+)anti-BPDE was bound to this deoxyoligomer duplex compared to the least reactive deoxyoligomer d(TAGTCGACTA). When the quantity of (+)anti-BPDE bound to dG was examined, most of the deoxyoligomers with two consecutive dG's reacted more extensively with (+)anti-BPDE than those which did not have two consecutive dG's. There was a good correlation between the sequence-selective binding of (+)anti-BPDE to self-complementary oligodeoxyribonucleotide duplexes and the sites of mutation induction reported in the literature. A modification of the $\sp{35}$S-postlabelling assay was developed to quantitate the amount of (+)anti-BPDE bound to each specific dG. When the quantity of (+)anti-BPDE bound to each specific dG was determined, preferred sites of binding of (+)anti-BPDE were found. A central dG surrounded on the 5$\sp\prime$ and 3$\sp\prime$ sides by dC reacted the most extensively: over four-fold more (+)anti-BPDE bound to this central dG compared to the least reactive central dG (AGT). It was also shown that binding to a central dG was preferred when a dG neighbor was present; no preference for a 5$\sp\prime$ over a 3$\sp\prime$ dG neighbor was found. Alternating pyrimidine-purine sequences were also found to enhance the binding of (+)anti-BPDE to a central dG. To further define the sequence-selective interactions of (+)anti-BPDE with oligodeoxyribonucleotides, this hydrocarbon diol epoxide was reacted with deoxyoligomer duplexes which contained varying lengths of dG runs. A greater quantity of (+)anti-BPDE bound per dG in the deoxyoligomer duplex containing 5 consecutive dG's compared to the duplex with 4 consecutive dG's. However, no differences in binding of (+)anti-BPDE per dG were found between the duplex containing 5 consecutive dG's compared to the duplex with 3 consecutive dG's. Moreover, it was also found that breaking a dG run of 3 by replacing the central dG with deoxycytidine had no effect on the quantity of (+)anti-BPDE which bound. The data presented in this thesis suggests that factors other than the primary sequence of nucleotides may be responsible for the binding of (+)anti-BPDE to runs of dG's. These studies indicate that base sequence has a major effect on the interaction of (+)anti-BPDE with oligodeoxyribonucleotide duplexes.

Degree

Ph.D.

Advisors

Baird, Purdue University.

Subject Area

Biochemistry

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