Abstract
Type II topoisomerases manage DNA supercoiling and aid chromosome segregation using a complex, ATP-dependent duplex strand passage mechanism. Type IIB topoisomerases and their homologs support both archaeal/plant viability and meiotic recombination. Topo VI, a prototypical type IIB topoisomerase, comprises two Top6A and two Top6B protomers; how these subunits cooperate to engage two DNA segments and link ATP turnover to DNA transport is poorly understood. Using multiple biochemical approaches, we show that Top6B, which harbors the ATPase activity of topo VI, recognizes and exploits the DNA crossings present in supercoiled DNA to stimulate subunit dimerization by ATP. Top6B self-association in turn induces extensive DNA bending, which is needed to support duplex cleavage by Top6A. Our observations explain how topo VI tightly coordinates DNA crossover recognition and ATP binding with strand scission, providing useful insights into the operation of type IIB topoisomerases and related meiotic recombination and GHKL ATPase machineries.
Highlights
The appropriate control of transcription, DNA replication, and chromosome segregation are essential to cell proliferation
Topo VI is a distributive DNA relaxase that preferentially recognizes DNA crossings We began our investigations of type IIB topoisomerase mechanism by measuring the affinity of M. mazei topoisomerase VI (topo VI) for DNAs of varying length or topological status
Using a broad range of functional and reporter assays, we show here that type IIB topoisomerases preferentially engage the DNA crossings and bends of supercoiled substrates, and that binding to supercoiled DNA in turn stimulates the nucleotide-dependent dimerization of Top6B and couples this movement to DNA cleavage and strand passage at a distance in Top6A
Summary
The appropriate control of transcription, DNA replication, and chromosome segregation are essential to cell proliferation. The catalytic domains that comprise the DNA breakage-reunion region of type IIA and IIB enzymes have been extensively shuffled One consequence of this rearrangement is that Top6A lacks a third subunit-subunit interface present in the type IIA enzymes, the ‘C-gate’ dimerization domain, which is thought to help mitigate the risk of aberrant double-strand break formation (Bates et al, 2011; Berger et al, 1996; Nichols et al, 1999; Roca, 2004). Our results explain why type IIB topoisomerases absolutely depend upon the ATPase activity of the B-subunit to generate double strand breaks These observations in turn reinforce the functional importance for DNA bending and potential T-segment-sensing elements in the related type IIA topoisomerases, and provide insights as to how recently discovered meiotic Top6B homologs might promote Spo mediated strand scission during meiotic recombination
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