We propose to continue and extend our studies on the formation, properties and resolution of Holliday junctions (HJs). The system we are using, the Int-dependent site-specific pathway of bacteriophage lambda, has been extensively studied at both the genetic and biochemical levels. It belongs to a large family of proteins from archaebacteria, eubacteria, mitochondria and yeast that catalyze rearrangements, in the absence of high energy cofactors, between DNA sequences with minimal homology to each other. Recombinases from this family have the unique capacity to both generate and resolve HJs. The Holliday junction is an important intermediate in """"""""DNA metabolism"""""""" and an understanding of its properties should contribute to the body of basic knowledge that serves as a platform for health-related advances in the general area of genetics. The proposed experiments depend upon our recent findings regarding the mechanisms of HJ formation and isomerization during site-specific recombination. Some of these recent insights have enabled the design of synthetic substrates that figure prominently in this proposal because they specifically monitor the dynamics of the ephemeral isomerization step. Six specific questions are addressed in this proposal: 1) What protein-DNA interactions govern resolution of the Holliday junction? 2) What protein-protein interactions influence the resolution reaction? 3) What dynamic features are important in the formation and resolution of Holliday junctions and what do they depend upon? a) Is ligation of the first strand-exchange a prerequisite (trigger) for HJ isomerization? b) Do the """"""""facilitating"""""""" (chemically uninvolved) Ints mimic (substitute for) those Ints that would have generated the HJ? c) Which specific protein-protein interactions play a role in moving the HJ """"""""forward"""""""" from one isomeric form to the other? d) Is the regulation of directionality exerted through the isomerization step?
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