Abstract:
Type IIA topoisomerase (Top2) regulates DNA topology by catalyzing a duplex-passage reaction through a transiently formed and enzyme-bridged reversible DNA break. The formation of Top2 DNA cleavage/religation center is driven by G-segment DNA-induced changes in protein tertiary and quaternary structure. These conformational changes, which involve repositioning of the TOPRIM domain with respect to the WHD and Tower domain, ensure key catalytic modules, such as the active site tyrosine and a divalent metal ion, are optimally placed and oriented around the scissile phosphate for executing reversible transesterification. For a more detailed understanding of the structural basis underlying the assembly of Top2’s DNA cleavage/religation center, we have determined the crystal structures of the DNA-binding/cleavage core of human Top2α and Top2β in a presumably on-pathway, DNA-binding competent state, in which the G-segment binding groove is fully exposed, ready for accepting an incoming DNA. Structural superimposition revealed how DNA-induced conformational changes associated with G-segment-binding lead to the formation of DNA cleavage center. Importantly, a novel type of protein-DNA interaction formed between the G-segment and a previously unrecognized yet strictly conserved structural module embedded within the TOPRIM domain was found to contribute significantly to Top2 function. Moreover, we performed a structure-guided biochemical assay to show how the function of a catalytic dead Top2 mutant can be restored by the additional of a small-molecule compound.
Keywords – type II topoisomerase, DNA cleavage and relegation, protein crystallography,
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