Structural and functional studies of the bacterial RecA protein

by 1976- Rajan, Rakhi

Double stranded (ds) DNA breaks are among the most detrimental types of DNA damage. dsDNA breaks can be repaired in cells by a process called homologous recombination. RecA is the key player that mediates the DNA strand exchange reaction in the recombination process. The gram positive bacterium Deinococcus radiodurans (Dr) is extremely resistant to high doses of ionizing radiation and thus of great interest for studying biological DNA repair processes and is of potential use in the bioremediation of radioactive waste. The resistance of Dr to extreme doses of ionizing radiation depends on 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 remarkable DNA repair capacity of Dr and the novel mechanistic features of its RecA protein, the x-ray crystal structure of Dr RecA in complex with ATP?S was determined at 2.5Å resolution. Like RecA from E. coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67Å. 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. Unique amino acid ii residues in Dr RecA cluster around a flexible ?-hairpin that has also been implicated in DNA binding. The details of Dr RecA structure are discussed in chapter 2. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5’-GCTGGTGG-3’). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. In chapter 3, a RecA coprotease assay was used to further probe the ssDNA sequence specificity of E. coli RecA protein. The extent of selfcleavage of a ?-repressor protein fragment in the presence of RecA, ADP-AlF4, and 64 different trinucleotide-repeating 15-mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. 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. The implications with regard to the biological mechanism of RecA are discussed. The inverse strand exchange pathway of Dr RecA was proposed based on in vitro strand exchange reactions, which gives an indirect measurement of the RecA-DNA iii interaction. The crystal structure of Dr RecA showed features consistent with the inverse strand exchange mechanism. In chapter 4, a set of experiments was designed to directly measure the interactions of Dr and Ec RecA proteins with ssDNA and dsDNA substrates. The experiments do not reveal any distinctive differences in the DNA-binding properties of the two proteins that are consistent with the proposed model for the inverse strand exchange pathway of Dr RecA.