Structure of a Synaptic γδ Resolvase Tetramer Covalently Linked to Two Cleaved DNAs

Abstract

The structure of a synaptic intermediate of the site-specific recombinase gammadelta resolvase covalently linked through Ser10 to two cleaved duplex DNAs has been determined at 3.4 angstrom resolution. This resolvase, activated for recombination by mutations, forms a tetramer whose structure is substantially changed from that of a presynaptic complex between dimeric resolvase and the cleavage site DNA. Because the two cleaved DNA duplexes that are to be recombined lie on opposite sides of the core tetramer, large movements of both protein and DNA are required to achieve strand exchange. The two dimers linked to the DNAs that are to be recombined are held together by a flat interface. This may allow a 180 degrees rotation of one dimer relative to the other in order to reposition the DNA duplexes for strand exchange.

PR65, the HEAT-repeat scaffold of phosphatase PP2A, is an elastic connector that links force and catalysis

“…The mechanical effects observed for PP2A are different from those previously described or proposed in mechanosensory systems (e.g., 4,5,35,41,50,51,59; SI Text, paragraph 5) with respect to the specific nature of the relevant motions. Moreover, in PP2A the conformational changes involved are mediated, reversibly, by an elastic connector.…”

Predicting Knot or Catenane Type of Site-Specific Recombination Products

“…This is supported by a recent crystal structure of a synaptic tetramer of γδ resolvase covalently catenated to two cleaved DNA molecules, indicating a subunit rotation of 180 • . 39 Additional experiments involving Tn3, Hin and Gin lends credence to the idea that each round of processive recombination acts identically. 2,48,63 For example, Heichman et al demonstrate that there are multiple rounds of exclusively clockwise subunit rotation of one set of Hin subunits after DNA cleavage.…”

“…17 With serine recombinases, DNA binding involves the conserved H-T-H domain, and a DNA binding domain on the C or N terminus of the protein. 39,44,54 For both families, there are also significant protein-protein interfaces with the other proteins in an assembled tetrameric complex. Additionally, DNA itself has a geometric diameter of 2nm, and, depending on the ionic conditions, a much greater electrostatic diameter (e.g.…”

“…[52][53][54] Furthermore, structures of two intermediate complexes -a synaptic γδ resolvase tetramer covalently linked to two cleaved DNAs, and the Flp recombinase-Holliday junction complex have at most one crossing between sites and none within a site. 39,42 Thus the large-scale conformational changes necessary to unwrap crossings during the reaction imply it is unlikely that the crossover sites contain additional crossings at synapsis or that a productive synapse does not exist.…”

“…with γδ and Tn3 resolvase), or inside the 4 recombinase subunits. [36][37][38][39][40][41][42][43] We will use the term recombinase-DNA complex to refer to B together with the substrate. If the recombinase complex meets the substrate in precisely the two crossover sites then we say the recombinase complex is a productive synapse.…”