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Design and chemical synthesis of sequence specific DNA cleaving metalloproteins

Citation

Mack, David Phillip (1991) Design and chemical synthesis of sequence specific DNA cleaving metalloproteins. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-06282007-112041

Abstract

Part I: Affinity Cleaving Studies of the DNA Binding Domain of Hin Recombinase.

Previous studies have shown that attachment of ethylenediaminetetraacetic acid (EDTA) to the NH2 terminus of the DNA binding domain of Hin recombinase creates an affinity cleaving molecule. The DNA cleavage pattern produced by this molecule has allowed a model for the DNA binding domain to be put forward which includes the NH2 terminus binding in the minor groove and a helix-turn-helix structure binding in the major groove. In this work the affinity cleaving methodology has been extended by placing the EDTA on a lysine side chain. Differential protection of the [alpha]- and [epsilon]- amino groups of lysine has allowed EDTA to be attached at the COOH terminus and at an internal position of the protein. Using these techniques EDTA has been attached at the COOH and NH2 terminus of the recognition helix of the DNA binding domain of Hin recombinase. Affinity cleaving studies with these molecules allows the orientation of the recognition helix on the DNA to be determined, as well as refine our model of the protein-DNA complex. The techniques developed are general and offer a powerful tool for examining the nature of protein-DNA complexes in the absence of crystallographic or NMR data.

Part II: Design of a DNA Cleaving Protein Consisting of Only Natural Amino Acids.

Attachment of the metal binding tripeptide, Gly-Gly-His, to the amino terminus of the DNA binding domain of Hin recombinase (residues 139-190) creates a new 55 residue protein containing only naturally occurring [alpha]-amino acids, GGH(Hin 139-190), with two structural domains each with distinct functions, sequence-specific recognition and cleavage of double helical DNA. This protein has been shown by footprinting to be competent to bind at four Hin sites, each 13 base pairs in length. In the presence of Cu(II), hydrogen peroxide and sodium ascorbate strong cleavage of the DNA occurs at one of the four sites by oxidative degradation of the deoxyribose backbone. Further, in the presence of Ni(II) and an oxygen atom source (eg. monoperoxyphthalic acid), the sequence specificity and efficiency of the DNA cleavage are remarkably altered. The nickel-mediated cleavage occurs at all four binding sites, is more rapid and efficient, and requires only one equivalent of an oxygen atom donor. At the hixL site, cleavage occurs predominantly at a single deoxyribose position on one strand of each binding site. Studies show that modification of the ligand system, Gly-Gly-His, can have tremendous effects on the intensity and position of DNA cleavage, and further, that for DNA cleavage mediated by the Ni(II)-protein a free amino terminus is required. Mechanistic studies indicate that the DNA cleavage by Ni(II)•GGH(Hin 139-190) likely results from abstraction of the C-4' hydrogen atom from the deoxyribose backbone by a high valent nickel-oxo species to produce a base labile modification. The tripeptide Gly-Gly-His thus is a metal specific structural domain with the function of substrate directed oxidation.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Unknown, Unknown
Defense Date:14 August 1990
Record Number:CaltechETD:etd-06282007-112041
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-06282007-112041
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:2764
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:23 Jul 2007
Last Modified:26 Dec 2012 02:54

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