Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

Significance of Knotted Structures for Function of Proteins and Nucleic Acids

Saturday Abstracts

Structural Basis for Regulation of Site-specific DNA Recombinases by DNA Topology Phoebe Rice 1 , Sherwin P. Montano 1 , Sally J. Rowland 2 , James R. Fuller 1 , Martin R. Boocock 2 , W. Marshall Stark 2 . 1 The University of Chicago, Chicago, USA, 2 University of Glasgow, Glasgow, United Kingdom. DNA resolvases are a group of site-specific recombinases that are regulated by DNA topology, and that act to “resolve” a circular DNA molecule into two smaller ones. We study two related resolvases: 1) Sin, which is encoded by many staphylococcal plasmids and presumably aids in their stability by resolving dimeric forms into monomeric ones and 2) Tn3, which is encoded by the Tn3 transposon and resolves the fused-circle cointegrate product of the replicative transposition process into two product circles. These resolvases are only catalytically active when incorporated into a larger complex (a “synaptosome”) that brings together the two DNA partners and traps 3 supercoiling nodes (dsDNA over dsDNA crossings) between them. Rendering activity dependent on synaptosome formation serves several purposes. 1st, it favors intra- over inter-plasmid recombination because trapping 3 crossings is easier within one supercoiled circle than between two separate ones. 2nd, it ensures correct alignment of the two crossover sites (parallel vs. antiparallel). 3rd, when the strand exchange process is carried out within the synaptosome it relaxes supercoiling strain and is thus energetically favorable. We used a combination of crystallography, SAXS and molecular biology to construct 3D models for the both the Sin and Tn3 resolvase synaptosomes. For both systems, individual structures of all the sub-complexes can be docked together in silico in a manner that agrees with activity data on a large array of mutant and chimeric proteins with altered DNA- and protein-protein interactions. Our results show that even though protein sequence and mechanistic similarities show these recombinases must have diverged from a common ancestor, they probably later converged upon the use of a synaptic complex that traps 3 supercoiling nodes because it is so well-suited to channeling the reaction to produce only resolution products.

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