Catalytic mechanism of DNA topoisomerase IB

Type IB topoisomerases and tyrosine recombinases are structurally homologou s strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme inter mediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-2 23, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 8'-bridging phosphorothio late-modified DNAs to implicate Lys-167 as a general acid catalyst. The low er pK(a), of the 5'-S leaving group versus 5'-O restored activity to the K1 67A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts i nto the minor groove upon DNA binding. Coupling of conformational changes i n a general acid loop to covalent catalysis of phosphoryl transfer is one o f several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.