Structural and functional studies of the bacterial RECA protein

by Rajan, Rakhi

Abstract (Summary)
RecA is the key player that mediates the DNA strand exchange reaction in the recombination process, which is essential for the repair of double stranded (ds) DNA breaks. The bacterium Deinococcus radiodurans (Dr) is extremely resistant to high doses of ionizing radiation because of its highly efficient capacity to repair dsDNA breaks. The Dr RecA protein promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA. In order to gain insight into the novel mechanistic features of Dr RecA protein, the x-ray crystal structure of Dr RecA was determined. Although the overall fold of Dr RecA is similar to E. coli RecA, there is a large reorientation of the C-terminal domain, which in E. coli RecA has a site for binding dsDNA. Compared to E. coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences. The binding of RecA to DNA is phased relative to the GT sequences, with a periodicity of three nucleotides. A RecA coprotease assay was used to further probe the ssDNA sequence specificity of E. coli RecA protein. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. S-Ribosylhomocysteinase (LuxS) is the enzyme which catalyses the synthesis of the precursor of type II bacterial quorum sensing molecule (AI-2). AI-2 is very important in antibiotic development, since it mediates inter-species communication with in bacteria. A catalytically inactive mutant of Bacillus subtilis LuxS was co-crystallized with a 2-ketone intermediate. The structure reveals that the C2 carbonyl oxygen is directly coordinated with the metal ion, providing strong support for the proposed Lewis acid function of the metal ion during catalysis.
Bibliographical Information:


School:The Ohio State University

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:homologous recombination reca double stranded dna break x ray crystallography luxs


Date of Publication:01/01/2007

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