University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Summer 2010 Requirements for Catalysis in Cre Recombinase Bryan P S Gibb Biochemistry and Molecular Biophysics, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Gibb, Bryan P S, "Requirements for Catalysis in Cre Recombinase" (2010). Publicly Accessible Penn Dissertations. 427. https://repository.upenn.edu/edissertations/427 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/427 For more information, please contact [email protected]. Requirements for Catalysis in Cre Recombinase Abstract Cre recombinase, a member of the tyrosine recombinase (YR) family of site-specific ecombinasesr 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 ecombination,r 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. eCr 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. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Biochemistry & Molecular Biophysics First Advisor Dr. Gregory Van Duyne Keywords Cre, recombination, site-specific, type IB opoisomert ase, tyrosine recombinase Subject Categories Biochemistry, Biophysics, and Structural Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/427 ACKNOWLEDGMENTS I owe a great deal of gratitude to my family, friends, co-workers, and Dr. Greg Van Duyne. This dissertation would not have been possible without their help, advice and support. Greg has been a fantastic mentor whom I will always look up to (figuratively speaking). While in the lab I have compiled a power list of skills at the bench and learned valuable lessons in critical thinking and reasoning. Greg allowed me freedom to explore all of my crazy ideas even though I'm sure he knew that many of them would fail. I have been fortunate to work in his lab and I greatly appreciate the opportunity as I am a better scientist for it. I would like to thank the past and present members of the Van Duyne lab for their constant support and discussions, science related or not. I would specifically like to thank Dr. Kaushik Ghosh and Dr. Kushol Gupta for teaching me 'the ropes' of Cre biochemistry and crystallization. I had many helpful scientific discussions with Dr. Kay Perry, Dr. Peng Yuan, Dr. Karen Rutherford, and Renne Carrington. James Chen helped with Cre assays. I have made many friends while at Penn, but would especially like to acknowledge Dr. David Hokanson, Dr. Bob Daber, and Tammer Farid. Without Dave, I would not have chosen to attend graduate school at UPenn, or taken up brewing beer as a hobby. Bob and I commuted from New Jersey for several years and was a constant source of support. Tammer, aside from providing a place for me to live for a year has been a great friend and instigator in acquiring a hobby for working on cars. II To my family, thank you for always being supportive. To my lovely wife Jennifer: You have been with me through all of this. Without your unwavering support I would not be here today. Thank you so much for your patience and understanding. To my parents, thank you for being a constant source of motivation by never failing to ask me 'so when do you think you are going to finish?' I must also thank my thesis committee, Dr. Mitch Lewis, Dr. Ronen Marmorstein, Dr. Kate Ferguson, and Dr. Kim Sharp for their input. III ABSTRACT REQUIREMENTS FOR CATALYSIS IN CRE RECOMBINASE Bryan P.S. Gibb Gregory Van Duyne, Ph.D., Thesis Advisor 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 IV 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. V Table of Contents Chapter 1. Introduction....................................................................................1 1.1 Site specific recombination...................................................................................................1 1.2 Tyrosine recombinase family members................................................................................5 1.2.1 Type Ib topoisomerases................................................................................................6 1.2.2 Cre and Flp: Simple YRs..............................................................................................7 1.2.3 λ-int and XerCD: Complex YRs....................................................................................8 1.3 Recombinases as tools for DNA manipulation....................................................................11 1.4 Mechanism of recombination..............................................................................................12 1.4.1 Phosphoryl-transfer chemistry....................................................................................12 1.4.2 Architecture.................................................................................................................19 1.5 Thesis objectives.................................................................................................................24 Chapter 2. Requirements for catalysis in the Cre active site (adapted from Gibb, B. et al. Nucleic Acids Research. 2010.)................................................26 2.1 Introduction.........................................................................................................................26 2.2 Structure of a Cre-loxP transition state