Topoisomerases are enzymes that resolve chromosome entanglements and control DNA superhelicity during transcription and replication. Topoisomerase VI (topo VI) is a type IIB topoisomerase found in archaea and plants that catalyzes the ATP-dependent transport of one DNA duplex through a transient, enzyme-mediated break in another. Topo VI is evolutionarily distinct from the type IIA topoisomerases employed by eukaryotes and bacteria, but is the ancestor of the Spo11 core complex, which cleaves duplex DNA to promote meiotic recombination. In the present proposal, I seek to answer longstanding questions about how topo VI engages DNA and how the enzyme?s structure and response to ligand binding are paralleled by the Spo11 core complex.
Aim 1 will address how topo VI binds either one or two DNA segments as part of its reaction cycle. Biochemical approaches have indicated that the binding mechanism for DNA, and the regulation of topo VI ATPase activity, require both DNA bending and recognition of a crossover formed between cleavage- and transport-DNA segments. The physical basis for these interactions will be established by defining the atomic-level details of distinct topo VI-DNA bound states using 3D cryo-EM.
Aim 2 will focus on the Spo11 core complex. Analysis of the homology between topo VI and components of the Spo11 core complex has raised questions as to the extent to which the mechanism of DNA binding and DNA cleavage regulation is related between the two systems. To date, the overall structure of the Spo11 core complex is unknown, as is the manner by which its subunits (which include Ski8, Rec102, and Rec104 in budding yeast) interact. I will determine the structural organization of the core complex, define subunit interaction points, and determine the structural response of the complex to binding ligands such as DNA. SAXS will be used to develop a 3D envelope of the complex and determine whether and how this shape changes in the presence of DNA and nucleotide; negative-stain EM will be used concurrently to spatially localize subunits and determine a 3D model of the core complex. The outcomes of this study will have a number of sustained impacts on the field, from providing the first views of how type IIB family topoisomerases/nucleases engage DNA to establishing how ATP influences DNA break formation by these enzymes. From a human health perspective, the failure to properly form DNA double-strand breaks during meiotic recombination results in chromosome nondisjunction at the first meiotic division and consequently birth defects and developmental disorders. The proposed work will have a significant impact on understanding the molecular basis by which Spo11 and its partners collaborate to control the initiation of meiotic recombination, providing a molecular explanation for how mutations in the core complex can disrupt this process, possibly leading to male infertility, developmental disorders, and genomic instability in cancer.
The proper resolution of chromosome entanglements is essential to cell viability; genetic recombination is additionally a crucial juncture in sexual reproduction at which chromosome errors can be produced. Recombination is initiated by an assembly of different protein molecules, several of which are evolutionarily related to a molecular machine called topoisomerase VI that disentangles DNA. This proposal aims to understand the mechanisms by which both systems respond to and act on target DNA segments, thereby contributing to fundamental knowledge about how assemblies of proteins and nucleic acids control the flow of genetic information.
