Date of Award

1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Kathleen M. Morden

Abstract

Mismatched base pairs have been implicated in mutations and arise from transitions or transversions of the base pairs during replication. An oligonucleotide duplex containing adjacent G$\cdot$A and A$\cdot$A mismatches, d(GCGAAT)$\cdot$d(ATAAGC), has been investigated using molecular mechanics and dynamics. Experimental NMR data obtained for a related molecule was used to constrain the model which suggested an unusual hydrogen-bonding for both mismatches. Between the G$\cdot$A and A$\cdot$A mismatches, defined by the unusual base pairing, there is a large twist angle. The remainder of the base pairs are B-DNA conformations. The effect of an unpaired base on the local geometry of an oligonucleotide duplex containing an (oligo dA)$\cdot$(oligo dT) tract was investigated using molecular modeling. The following duplexes have been studied: $$\vbox{\halign{#\hfil&&\qquad#\hfil\cr &dGCGAA{\bf X}AAGCG &dGCGAA-AAGCG &dGCGAAAACGC\cr &\ CGCTT-TTCGCd &CGCTT{\bf Y}TTCGCd &CGCTTTTCGCd\cr}}$$where X is cytosine, thymine or guanine, and where Y is guanine, adenine or thymine. The structures are of interest because they contain an A-tract, which is implicated in DNA bending and a region that is a model for mutational hot spots. The models were built with the unpaired base in two possible orientations, extrahelical and intrahelical. The results indicate that in the intrahelical conformation the flanking base pairs form a wedge and in the extrahelical conformation the flanking base pairs remain in a B-DNA stack. The extrahelical pyrimidines are in the minor groove whereas the extrahelical purines have two possible conformations either in the major groove or in the minor groove. The effect of an unpaired base on the base-pair opening rate of an oligonucleotide duplex containing an (oligo dA)$\cdot$(oligo dT) tract has been investigated using proton exchange NMR techniques on: $$\vbox{\halign{#\hfil&&\qquad#\hfil\cr &dGCGAA{\bf \ G}AAGCG &dGCGAA-AAGCG &dGCGAAAACGC\cr &\ CGCTT-TTCGCd & CGCTT{\bf \ G}TTCGCd &CGCTTTTCGCd\cr}}$$For d(GCGAAGAAGCG)$\cdot$d(CGCTTTTCGC) the base pairs adjacent to the unpaired G have faster opening rates than do those base pairs further away. For d(GCGAAAAGCG)$\cdot$d(CGCTTGTTCGC) the base pairs adjacent to the unpaired base have very fast opening rates. The activation energies are similar for d(GCGAAAAGCG)$\cdot$d(CGCTTTTCGC), d(GCGAAGAAGCG)$\cdot$d(CGCTTTTCGC) and d(GCGAAAAGCG)$\cdot$d(CGCTTGTTCGC).

Pages

299

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