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Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA

Maher, L. James, III and Dervan, Peter B. and Wold, Barbara J. (1990) Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA. Biochemistry, 29 (37). pp. 8820-8826. ISSN 0006-2960. doi:10.1021/bi00489a045.

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Pyrimidine oligonucleotides recognize extended purine sequences in the major groove of double-helical DNA by triple-helix formation. The resulting local triple helices are relatively stable and can block DNA recognition by sequence-specific DNA binding proteins such as restriction endonucleases. Association and dissociation kinetics for the oligodeoxyribonucleotide 5'-CTCTTTCCTCTCTTTTTCCCC (bold C's indicate 5-methylcytosine residues) are now measured with a restriction endonuclease protection assay. When oligonucleotides are present in greater than 10-fold excess over the DNA target site, the binding reaction kinetics are pseudo first order in oligonucleotide concentration. Under our standard conditions (37 degrees C, 25 mM Tris-acetate, pH 6.8, 70 mM sodium chloride, 20 mM magnesium chloride, 0.4 mM spermine tetrahydrochloride, 10 mM beta-mercaptoethanol, 0.1 mg/mL bovine serum albumin) the value of the observed pseudo-first-order association rate constant, k_(2obs), is 1.8 x 10^3 +± 1.9 x 10^2 L.(mol of oligomer^-1·s^-1. Measurement of the dissociation rate constant yields an equilibrium dissociation constant of approximately 10 nM. Increasing sodium ion concentration slightly decreased the association rate, substantially increased the dissociation rate, and thereby reduced the equilibrium binding constant. This effect was reversible by increasing multivalent cation concentration, confirming the significant role of multivalent cations in oligonucleotide-directed triple-helix formation under these conditions. Finally, a small reduction in association rate, a large increase in dissociation rate, and a resulting reduction in the equilibrium binding constant were observed upon increasing the pH between 6.8 and 7.2.

Item Type:Article
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Dervan, Peter B.0000-0001-8852-7306
Wold, Barbara J.0000-0003-3235-8130
Additional Information:© 1990 American Chemical Society. Received March 21, 1990; Revised Manuscript Received May 25, 1990. Supported by research grants from the National Institutes of Health (GM-35724) and the Beckman Institute of the California Institute of Technology and an American Cancer Society Postdoctoral Fellowship to L.J.M.
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Caltech Beckman InstituteUNSPECIFIED
American Cancer SocietyUNSPECIFIED
Issue or Number:37
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Official Citation:Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA L. James Maher III, Peter B. Dervan, and Barbara J. Wold Biochemistry 1990 29 (37), 8820-8826 DOI: 10.1021/bi00489a045
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ID Code:66957
Deposited By: Victoria Brennan
Deposited On:19 May 2016 22:16
Last Modified:11 Nov 2021 00:24

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