SITE-DIRECTED MUTAGENESIS OF CONSERVED ASPARTATES, GLUTAMATES AND ARGININES IN THE ACTIVE-SITE REGION OF ESCHERICHIA-COLI DNA TOPOISOMERASE-I

To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a c ovalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage, Strand passage then takes place to change the linkin g number, This is followed by DNA religation during which the displace d DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond, Mg(II) is required for the relaxation activi ty of type LA and type II DNA topoisomerases, A number of conserved am ino acids with acidic and basic side chains are present near Tyr-319 i n the active site of the crystal structure of the 67-kDa N-terminal fr agment of Escherichia coil DNA topoisomerase I, Their roles in enzyme catalysis were investigated by site-directed mutation to alanine, Muta tion of Arg-136 abolished all the enzyme relaxation activity even thou gh DNA cleavage activity was retained, The Glu-9, Asp-lll, Asp-113, Gl u-115, and Arg-321 mutants had partial loss of relaxation activity in vitro, All the mutants failed to complement chromosomal topA mutation in E. coil AS17 at 42 degrees C, possibly accounting for the conservat ion of these residues in evolution.