Structural studies of viral and eukaryotic type I DNA topoisomerases

摘要

DNA topoisomerases are enzymes that control the topological properties of DNA inside a cell or virion. These enzymes are ubiquitous and carry there topoisomerase activity by transiently breaking one or both strands of DNA, and passing the strand(s) through a protein gate. These molecules are therefore involved in essential cellular processes like supercoil relaxation, recombination, transcription, and DNA replication. Eukaryotic-like DNA topoisomerases I comprise all the topoisomerases encoded by eukaryotic cellular organisms, and poxviral family of topoisomerases. Prototypical members of the eukaryotic cellular family and the poxviral family are the S. cerevisiae and the vaccinia virus DNA topoisomerases. These enzymes relax both positively and negatively supercoiled DNA, and have been the target of extensive biochemical and therapeutic studies. This thesis describes purification, crystallization, X-ray structure elucidation, and atomic structure of the amino terminal 9 kDa domain of the vaccinia virus DNA topoisomerase I. It also describes the model building, model refinement, and structural analyses of the atomic structure of a 26 kDa domain of the S. cerevisiae DNA topoisomerase I. The 9 kDa amino terminal fragment of the vaccinia virus DNA topoisomerase is a five stranded anti-parallel $beta$ sheet structure that forms a compact hydrophobic core using connecting $alpha$ helices and loops. The structure has allowed to detail single site activity mutants of the intact topoisomerase, to map basic residues that directly interact with DNA, and to structurally compare it with the S. cerevisiae DNA topoisomerase 26 kDa domain. The latter is a two domain protein, connected by two V-shaped $alpha$ helices and an extended segment. Its protein architecture is unique, and biochemical and electrostatic calculations suggest a novel mode of DNA binding. The 9 kDa and 26 kDa domains from poxviral and eukaryotic cellular topoisomerases are structurally dissimilar and this suggests that the former forms a separate unique family of DNA topoisomerases that are much smaller than all other known topoisomerases and yet carry out identical reactions. These structures represent the first and only atomic level information available on eukaryotic-like DNA topoisomerases so far.