Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA

Human topoisomerase I helps to control the level of DNA supercoiling in cel ls and is vital for numerous DNA metabolic events, including replication, t ranscription, and recombination. The 2.6 Angstrom crystal structure of huma n topoisomerase I in noncovalent complex with a DNA duplex containing a cyt osine at the -1 position of the scissile strand rather than the favored thy mine is reported. The hydrogen bond between the O2 position of this -1 base and the is an element of-amino of the conserved Lys-532 residue, the only base-specific contact observed previously in the human topoisomerase I-DNA interaction, is maintained in this complex. Several unique features of this structure, however, have implications for the DNA-binding and active-site mechanisms of the enzyme. First, the ends of the DNA duplex were observed t o shift by up to 5.4 Angstrom perpendicular to the DNA helical axis relativ e to structures reported previously, suggesting a novel degree of plasticit y in the interaction between human topoisomerase I and its DNA substrate. S econd, 12 additional residues at the NH2 terminus of the protein (Trp-203-G ly-214) could be built in this structure, and they were found to pack again st the putative hinge region implicated in the clamping of the enzyme aroun d duplex DNA. Third, a water molecule was observed adjacent to the scissile phosphate and the active-site residues: the potential specific base charac ter of this solvent molecule in the active-site mechanism of the enzyme is discussed. Fourth, the scissile phosphate group was found to be rotated by 75 degrees, bringing Lys-532 into hydrogen-bonding distance of one of the n onbridging phosphate oxygens. This orientation of the scissile phosphate gr oup implicates Lys-532 as a fifth active-site residue, and also mimics the orientation observed for the 3'-phosphotyrosine linkage in the covalent hum an topoisomerase I-DNA complex structure. The implications of these structu ral features for the mechanism of the enzyme are discussed, including the p otential requirement for a rotation of the scissile phosphate group during DNA strand cleavage and covalent attachment.