A novel and unified two-metal mechanism for DNA cleavage by type II and IA topoisomerases.
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.
Schmidt
Bryan H
BH
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Burgin
Alex B
AB
Deweese
Joseph E
JE
Osheroff
Neil
N
Berger
James M
JM
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
D014443:Tyrosine; D004247:DNA; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II