Abstract
The proteins of SMC family are characterised 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 recN(K35D). Reverse genetics was utilized to delete the entire recN gene (Delta recN108) or introduce the recN(K35D) gene into the E. coli chromosomal DNA. The recN(K35D) cells showed decreasing in the frequency of excision of Tn10 from gal76::Tn10 after treatment with mitomycin C compared to recN(+) cells. The Delta recN108 cells showed an uninduced increase frequency of Tn10 excision from gal76::Tn10 in rec(+) background. While, recBC sbcBC Delta recN108 cells are completely deficient in Tn10 excision. The recombination proficiency is reduced in cells carrying recBC sbcBC cells in addition recN(K35D) 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 recN(K35D). (C) 2011 Production and hosting by Elsevier B.V. on behalf of King Saud University.