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Effects of the A·T/T·A Degeneracy of Pyrrole−Imidazole Polyamide Recognition in the Minor Groove of DNA

White, Sarah and Baird, Eldon E. and Dervan, Peter B. (1996) Effects of the A·T/T·A Degeneracy of Pyrrole−Imidazole Polyamide Recognition in the Minor Groove of DNA. Biochemistry, 35 (38). pp. 12532-12537. ISSN 0006-2960. http://resolver.caltech.edu/CaltechAUTHORS:20160510-102101250

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Abstract

Pairing rules have been developed to predict the sequence specificity of minor groove binding polyamides containing pyrrole (Py) and imidazole (Im) amino acids. An Im/Py pair distinguishes G·C from C·G and both of these from A·T/ T·A base pairs. A Py/Py pair appears not to distinguish A·T from T·A base pairs. To test the extent of this degeneracy, the affinity and binding orientation of the hairpin polyamide ImPyPy-γ-PyPyPy-β-Dp were measured for eight possible five base pair 5‘-TG(A,T)_3-3‘ match sites. Affinity cleavage experiments using a polyamide with an EDTA·Fe(II) moiety at the carboxy terminus, ImPyPy-γ-PyPyPy-β-Dp-EDTA·Fe(II), are consistent with formation of an oriented 1:1 hairpin polyamide complex at all eight 5‘-TG(A,T)_3-3‘ binding sites [20 mM HEPES, 200 mM NaCl, 50 mg/ml glycogen, pH 7.0, 22 °C, 5 mM DTT, 1 mM Fe(II)]. Quantitative DNase I footprint titration experiments reveal that ImPyPy-γ-PyPyPy-β-Dp binds all eight 5‘-TG(A,T)_3-3‘ target sites with only a 12-fold difference in the equilibrium association constants between the strongest site, 5‘-TGTTT-3‘ (K_a = 2.1 × 10^8 M^(-1)), and the weakest site, 5‘-TGAAT-3‘ (K_a = 1.8 × 10^7 M^(-1)) (10 mM Tris·HCl, 10 mM KCl, 10 mM MgCl_2, 5 mM CaCl_2, pH 7.0, 22 °C). This relatively small range indicates that the Py/Py pair is approximately degenerate for recognition of A,T base pairs, affording generality with regard to targeting sequences of mixed A·T/T·A composition.


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http://dx.doi.org/10.1021/bi960744iDOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi960744iPublisherArticle
Additional Information:© 1996 American Chemical Society. Received March 27, 1996; Revised Manuscript Received July 30, 1996. Publication Date (Web): September 24, 1996. This research was supported by National Institutes of Health Grant GM-27681, a National Science Foundation predoctoral fellowship to S.W., and a Howard Hughes Medical Institute predoctoral fellowship to E.E.B.
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Funding AgencyGrant Number
NIHGM-27681
NSF Predoctoral FellowshipUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Record Number:CaltechAUTHORS:20160510-102101250
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160510-102101250
Official Citation:Effects of the A·T/T·A Degeneracy of Pyrrole−Imidazole Polyamide Recognition in the Minor Groove of DNA Sarah White, Eldon E. Baird, and Peter B. Dervan Biochemistry 1996 35 (38), 12532-12537 DOI: 10.1021/bi960744i
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ID Code:66882
Collection:CaltechAUTHORS
Deposited By: Victoria Brennan
Deposited On:18 May 2016 21:42
Last Modified:18 May 2016 21:42

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