Investigating the Biochemical Functions of E.coli RecA Mutant F217Y

The bacterial DNA recombinase protein RecA is a well-known activator of the genetic response (known as SOS) induced by DNA damage. Regulation of SOS-induced DNA damage repair is essential to genomic stability. Previous research indicates that a mutation of the 217^th amino acid in the wild type RecA amino acid sequence (replacement of the amino acid phenylalanine (F) with the amino acid Tyrosine (Y), F217Y) results in a constitutive SOS response in E. coli cells.  It is important to investigate the biochemical functions of RecA F217Y and the roles this protein plays in genomic stability and the repair of DNA damage. Therefore, the RecA F217Y mutant RecA protein was purified to near-homogeneity and tested for hyperactive biochemical properties. Using a coupled-spectrophotometric assay, we find that RecA F217Y competes better for DNA sites with the SSB protein than the wild-type construct, a function critical to the regulation of SOS induction.  Additionally, DNA binding assays suggest that F217Y binds to undamaged DNA, whereas wild type RecA binds to damaged DNA.