Crystal Structure of a Bacterial Topoisomerase IB in Complex with DNA Reveals a Secondary DNA Binding Site

Abstract

Summary Type IB DNA topoisomerases (TopIB) are enzymes that relax supercoils by cleaving and resealing one strand of duplex DNA within a protein clamp that embraces a DNA segment. A longstanding conundrum concerns the capacity of TopIB enzymes to stabilize intramolecular duplex DNA crossovers and, in the case of poxvirus TopIB, form protein-DNA synaptic filaments. Here we report a structure of D. radiodurans TopIB in complex with a 12-bp duplex DNA that demonstrates a secondary DNA binding site located on the C-terminal domain. It comprises a distinctive interface with one strand of the DNA duplex and is conserved in all TopIB enzymes. Modeling of a TopIB with both DNA sites suggests that the secondary site could account for DNA crossover binding, nucleation of DNA synapsis, and generation of a filamentous plectoneme. In support of this, mutations of the secondary site eliminate synaptic plectoneme formation without affecting DNA cleavage or supercoil relaxation.

All tangled up: how cells direct, manage and exploit topoisomerase function

“…1D). Type IB proteins preferentially bind positively- or negatively- supercoiled substrates rather than relaxed substrates 17,18 using an interaction surface outside of its primary active site to bridge distal DNA segments 19 . Some variants show a proclivity for positively-supercoiled DNA, which they can relax at a faster rate 18 .…”

The dynamic interplay between DNA topoisomerases and DNA topology

“…Topo IB inhibitor-mediated DNA cleavage complex formation is enhanced by supercoiling of the DNA substrate (Seol et al 2015), although the relative enhancement is modulated by the chirality of supercoiling, the specific type of inhibitor, and the inherent chiral-dependence of cleavage (Gentry et al 2011;McClendon and Osheroff 2006;Seol et al 2015). This enhancement may arise from the higher affinity of Topo IB for supercoiled DNA (Madden et al 1995), possibly due to preferential binding at DNA crossovers (Patel et al 2010;Zechiedrich and Osheroff 1990); however, recent measurements performed at low Topo IB concentrations suggest there is little difference in affinity between relaxed and supercoiled DNA (Litwin et al 2015). Alternatively, enhanced Increased torsion also hinders and can reverse DNAwrapping interactions of nucleosomes.…”

Order from the Order: How a Spatiotemporal Genetic Program Is Encoded in a 2-D Genetic Map of the Bacterial Chromosome

“…Accordingly, computational simulations of the molecular dynamics of supercoiled DNA also suggest that under excessive torsional stress, the right-handed (nega- tive) supercoils are less stable than the left-handed coils [Harris et al, 2008]. The right-and left-handed crossovers thus not only have different stability but also distinct local geometries that can be specifically recognised by DNA topoisomerases -enzymes removing or adding the DNA coils and thus homeostatically adjusting the supercoiling level in optimising the function of the replication, transcription and DNA recombination machineries [Patel et al, 2010;Timsit, 2011;Zechiedrich and Osheroff, 1990;Zechiedrich et al, 2000]. Various 3-D DNA structures stabilised by particular states of superhelical density in turn affect the binding of regulatory proteins including the RNAP-σ initiation factors and the transcription factors [Bordes et al, 2003;Brázda et al, 2012;Drew and Travers, 1984;Hancock et al, 2013;Kusano et al, 1996;Ouafa et al, 2012;Schneider et al, 1997], such that ultimately the changes in superhelical density are directly transmitted to the gene regulatory machinery.…”