Extensive Bioinformatics Analyses Reveal a Phylogenetically Conserved Winged Helix (WH) Domain (Zτ) of Topoisomerase IIα, Elucidating Its Very High Affinity for Left-Handed Z-DNA and Suggesting Novel Putative Functions

Abstract

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, “bubbles”, R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the “B-Z-topoII hypothesis” and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments (“Z-flipons”) as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover “conformase” activity on given G(ate) B-DNA segments (“Z-flipins”), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents.

Flipons and small RNAs accentuate the asymmetries of pervasive transcription by the reset and sequence-specific microcoding of promoter conformation

“…The enzyme may directly engage Z-DNA. A recent bioinformatic analysis provided evidence for a winged helix-turn-helix Z-DNA binding domain present in TOP2A that is distinct from Zα ( 74 ). Interestingly, TOP2 also facilitates the folding of chromosomes to approximate enhancers and promoters, providing the framework for the assembly of the mediator complex that controls gene expression by integrating physiological and developmental signals within the cell (represented by MED12 in Fig.…”

“…More proteins that interact with Z-DNA and Z-RNA likely exist with variants that are causal for disease. The search for such proteins has not so far been exhaustive, but is underway ( 74 ).…”

Flipons: The discovery of Z-DNA and soft-wired genomes

“…The formation of Z-DNA may localize Z-DNA-binding proteins as well. Martin Bartas and Tom Jovin have recently modeled a Z-DNA-binding domain into a class of topoisomerase enzymes [129]. This particular version can cut both strands of a DNA segment to resolve the intertwining that occurs during replication of DNA and that arise because the new copies of DNA are intertwined.…”

No Time to Relax and Unwind: Exploration of Topoisomerases and a Growing Field of Study

“…Fourth, the integration of data from various sources has led to several studies that explore unknown or underappreciated aspects of topoisomerase function [19]. For instance, insights into the inter-relationships in the unstructured C-terminal domain may be explored using computational methods [20].…”