Site specific DNA recombination acts as a developmental switch in both prokaryotes and eukaryotes. The reaction can, in principle, rearrange genetic information via excision, inversion, or translocation, and control patterns of gene expression by turning promoters on or off. The basic chemistry of recombination involves phosphoryl transfer within or between nucleic acid chains, and must share mechanistic similarities with general recombination, RNA splicing and DNA transposition. This proposal seeks continued support for the PI's studies on the chemical and mechanistic features of strand breakage and reunion mediated by the Flp site-specific recombinase of S. cerevisiae. Flp is a member of the Integrase superfamily of recombinase proteins, which includes lambda integrase, Flp, the cre recombinase of P1 and the R recombinase of Zygosaccharomyces. These recombinases have limited sequence similarity within a 40 amino acid region in their carboxyl termini, with four invariant residues: two arginines, a histidine and a tyrosine. The tyrosine is in the active site of Flp and is the residue that becomes covalently linked to DNA by a 3'-phosphotyrosyl linkage. The Arg-His-Arg (RHR) triad appears to form part of the catalytic pocket as well. It is well established that a Flp monomer has only a partial active site. A complete active site is formed by sharing partial active sites of at least two monomers of the protein, one contributing the RHR triad, and the other the Tyr. The proposed experiments focus on three different issues related to the mechanism of the Flp mediated reactions.
The first aim i s to determine what is the oligomeric state of Flp that is catalytically relevant to each of the two steps of the reaction, strand breakage and reunion. The issue here is whether the recombinational unit of Flp is an asymmetric trimer as proposed by Qian and Cox or a tetramer composed of two asymmetric dimers as proposed by Lee and Jayaram. The proposed test is based on the demonstration that RHR triad mutants complement tyrosine mutants, i.e. that an active site can be assembled by a heterodimer of the two mutant proteins. The idea is to ask whether the resolution of a Holliday junction, which requires two active sites, can be carried out by a triad mutant in combination with a tyrosine mutant. If the active unit is an asymmetric trimer, then resolution should not be possible. Alternatively, if the active unit is a tetramer, then some resolution should occur. A further test involves constructing complexes with three active monomers and one Y343F monomer positioned at specific places and looking at the position of the resolution event.
The second aim i s to understand the mechanism of active site exclusion during Flp-mediated recombination. Flp recombination proceeds in two steps of single strand exchanges, implying that only two of the four potential active sites within a Flp tetramer are function at a time. The first pair of sites is used to form the Holliday junction intermediate, and the second pair to resolve it. The specific hypothesis to be tested is whether the DNA bend of >140 degrees that is induced by binding of two Flp monomers to a complete site is involved in this active site exclusion. The test involves building a set of Flp substrates containing bulges that would predispose the arms to stack in a bent form, and determine the asymmetry of cleavage by Flp. The substrates will be tested with wild type Flp and with mutant variants defective in DNA bending. The structure of the bulge-containing substrates will be confirmed by physical analysis, both NMR and FRET.
The third aim i s directed at understanding the role of DNA synapsis in both the initial strand cleavage reaction catalyzed by Flp and Holliday junction resolution event. The key question is whether substrate can enter the synaptic complex in a pre-cleaved state, or, alternatively, whether cleavage within a full site occupied by a Flp dimer is abortive, with a productive cleavage, capable of being resolved as a recombinant, only possible within a pair of synapsed full sites. The tests involve the building synthetic substrates to test both questions.
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