Genetic characterization of Escherichia coli RecN protein as a member of SMC family of proteins

The proteins of SMC family are characterized by having Walker A and B sites. The Escherichia coli RecN protein is a prokaryotic member of SMC family that involved in the induced excision of Tn10 and the repair of the DNA double strand breaks. In this work, the Walker A nucleotide binding site of the E. coli RecN protein was mutated by changing the highly conserved lysine residue 35 to the aspartic acid (D), designated as recNK35D. Reverse genetics was utilized to delete the entire recN gene (ΔrecN108) or introduce the recNK35D gene into the E. coli chromosomal DNA. The recNK35D cells showed decrease in the frequency of excision of Tn10 from gal76::Tn10 after treatment with mitomycin C compared to recN+ cells. The ΔrecN108 cells showed an un-induced increase in the frequency of Tn10 excision from gal76::Tn10 in rec+ background while, recBC sbcBC ΔrecN108 cells are completely deficient in Tn10 excision. The recombination proficiency is reduced in cells carrying recBC sbcBC cells in addition recNK35D mutation. We observed that the Walker A nucleotide binding site is important for the RecN protein. Strains that deleted recN gene are recombination deficient and more sensitive to mitomycin C than strains carrying recNK35D.