Date of Award

Summer 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Dr. Gregory Van Duyne

Abstract

Cre recombinase, a member of the tyrosine recombinase (YR) family of site-specific recombinases catalyzes DNA rearrangements using phosphoryl transfer chemistry that is identical to that used by the type IB topoisomerases (TopIBs). In this dissertation, the requirements for YR catalysis and the relationship between the YRs and the TopIBs are explored. I have analyzed the in vivo and in vitro recombination activities of all possible substitutions of the seven active site residues in Cre recombinase. To facilitate the interpretation mutant activities, I also determined the structure of a vanadate transition state mimic for the Cre-loxP reaction that allows for a comparison with similar structures from the related TopIBs. The results demonstrate that active site residues shared by the TopIBs are most sensitive to substitution. Two of the conserved active site residues in YRs have no equivalent in TopIBs. I have concluded that Glu176 and His289 in Cre evolved to have functional roles in site-specific recombination, that are unnecessary for relaxation by TopIB. His289 is not essential for cleavage of DNA, but accelerates water mediated hydrolysis of the 3'-phosphotyrosine covalent intermediate. Glu176 serves a structural role in facilitating the activation of Cre for cleavage by helping position the general acid, K201 in the active site. These residues may compensate for activity lost during the evolution of allosteric regulation required for recombination. Examination of two regions involved in protein:protein interactions in the Cre-DNA tetramer complex has led to the characterization of two modes of allosteric regulation. The first involves a C-terminal helix (hN), which binds in the pocket of a neighboring Cre monomer to serve as a regulatory switch by acting as a tether to position the tyrosine nucleophile (Y324). The second regulatory module involves the mobile β2-β3 hairpin carrying the general acid, K201. This hairpin must form synapsis dependent contacts to efficiently position K201 near O5' of the scissile phosphate. Cre has evolved to modulate the positions of critical residues for catalysis, which is an effective and highly sensitive mechanism of regulation of catalysis not shared by TopIBs.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS