Structural studies of homologous recombination in bacteria

by Xing, Xu

Abstract (Summary)
RecA is a central player in homologous recombination. The structures of E. coli RecA and its complex with ADP were solved 15 years ago, in which RecA is packed in a right-handed helical filament with a pitch of 82.7Å. In Chapter 2 the x-ray crystal structure of uncomplexed E. coli RecA in three new crystal forms is presented at resolutions of 1.9, 2.0, and 2.6Å respectively, with a relatively compressed pitch of ~74Å. The new structures are very similar to the original structure; however, they show significant variation in the C-terminal domain and the inter-filament packing interactions. In Chapter 3, the crystal structures of RecA in complex with MgADP and MnAMP-PNP are presented at resolutions of 1.9Å and 2.1Å, respectively, with a pitch of ~82Å. The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the ?-phosphate of AMP-PNP. Both structures are very similar to the original RecA structure. Based on the coprotease activity of RecA, AMP-PNP does not promote RecA into its active conformation, thus the crystal structure of the RecA-MnAMP-PNP complex most likely represents a preisomerization state of RecA protein that exists after ATP has bound, but before the conformational transition to the active state. The Red recombination system of bacteriophage ? catalyzes homologous recombination during the phage’s lytic growth stage in E. coli. ? protein, a component of the Red recombination, promotes a single-strand annealing reaction. In Chapter 4, a model for the domain structure and DNA binding regions of ? protein is presented based on limited protease digestion and biotinylation of lysine residues. ? protein consists of a N-terminal “core” domain that is resistant to proteases in the absence of DNA, a central region with enhanced resistance to proteases upon DNA complex formation, and a C-terminal domain that are sensitive to proteases in both the presence and absence of DNA. The N-terminal core and the central domain come together to bind DNA. A series of ? protein fragments have been purified and crystallized. The best crystal obtained so far diffracts to ~6Å.
Bibliographical Information:


School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:reca red? x ray crystallography limited protease digestion biotinylation domain structure


Date of Publication:01/01/2007

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