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Design and chemical synthesis of a sequence-specific DNA-cleaving metalloprotein: NI (II) GGH-γδ(141-183)


Graham, Kenneth Scott (1993) Design and chemical synthesis of a sequence-specific DNA-cleaving metalloprotein: NI (II) GGH-γδ(141-183). Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/7xmm-gb60.


Chapter I: Affinity-Cleaving Studies Examining the Orientation and Specificity of the DNA-Binding Domain from yδ Resolvase

The DNA binding domain of gd resolvase, residues 141-183, is thought to bind DNA by the helix-turn-helix motif based on sequence similarities with other known DNA binding proteins. Incorporation of the DNA cleaving moiety, EDTA•Fe at the amino and carboxy termini of yδ (141-183), allows the positions of these residue relative to the DNA bases at three resolvase binding sites, each consisting of inverted copies of an imperfectly conserved nine base pair consensus sequence, to be mapped by highresolution gel electrophoresis. Cleavage data for EDTA-yδ(141-183) reveal that the NH^2 terminus of the DNA binding domain of yδ resolvase is bound proximal to the minor groove of DNA near the center of the resolvase binding sites. Cleavage by EDTA•Fe attached to a lysine side chain at (Asn^183_Lys^183) at the COOH terminus of yδ (141-183) reveals that the putative recognition helix is in the adjacent major groove on the same face of the helix, oriented towards the center of the inverted repeats. Subsequent studies utilized affinity cleavage to analyze the effects of changes in amino acid composition of the recognition helix on the binding characteristics of yδ (141-183).In a systematic helix-switch experiment, the DNA contact residues of the putative recognition helix were exchanged for the DNA contact residues from another putative helix-turn-helix protein, Hin recombinase. Substitution of the amino acid in the putative recognition helix of yδ resolvase resulted in five proteins that showed no DNA binding specificity, six proteins that showed no significant alterations in DNA binding specificity, and four proteins that showed altered DNA binding specificity.

Chapter II: Design of a Sequence-Specific DNA-Cleavage Metalloprotein Ni(II) GGH- yδ (141-183)

A forty-six residue chymeric protein that combines the cuperic binding domain of serum albumin, GGH, with the DNA binding domain of yδ resolvase (residues 141-183), was synthesized. This protein, in the presence of Ni(II) and monoperoxyphthalic acid, cleaves DNA sequence specifically at one nucleotide adjacent to each of the six yδ resolvase binding sites contained within res. Cleavage occurs at the center of each of the dimeric binding sites, indicating that the position of the Ni(II)GGH is proximal to the minor groove. The characteristics of the DNA products created by Ni(GGH- yδ 141-183) cleavage of DNA indicates that cleavage is produced by some time of non-diffusible oxidizing species. ESR studies indicate that cleavage is not mediated by Ni(III)•GGH. Mechanistic studies indicate that DNA cleavage likely results from abstraction of the hydrogen atom at the C4' position of the deoxyribose backbone by some high valent, nickel-oxo species. Protocols for the synthesis of deuterated thymidines are included.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Gray, Harry B.
Defense Date:29 October 1992
Record Number:CaltechTHESIS:12182012-094906629
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7341
Deposited By: Dan Anguka
Deposited On:18 Dec 2012 18:27
Last Modified:09 Nov 2022 19:20

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