title: A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases.
abstract: Type II topoisomerases are required for the management of DNA tangles and supercoils, and are targets of clinical antibiotics and anti-cancer agents. These enzymes catalyse the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or 'gates'. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyse G-segment cleavage. Recent efforts have proposed that strand scission relies on a 'two-metal mechanism', a ubiquitous biochemical strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topoisomerase II covalently linked to DNA through its active-site tyrosine at 2.5A resolution, revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking experiments indicate that cleavage is catalysed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a molecular 'trapdoor' to prevent subunit dissociation during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
authors.forename: Bryan H
authors.initials: BH
authors.lastname: Schmidt
authors.forename: Alex B
authors.initials: AB
authors.lastname: Burgin
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Deweese
authors.forename: Neil
authors.initials: N
authors.lastname: Osheroff
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
mesh_terms: D000494:Allosteric Regulation; D001483:Base Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014443:Tyrosine
chemical_list: D014443:Tyrosine; D004247:DNA; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II