We propose to study the formation, properties and resolution of Holliday recombination intermediates using the Int-dependent site-specific pathway of bacteriophage lambda. Several of the specific aims are motivated, or will be facilitated, by the following results and observations from the previous project period: two families of Holliday junction suicide substrates were developed to: a) disengage Int cleavage events from the closely coupled ligation reactions and, b) to direct specific Int protomers to specific sites; two partner Ints cannot carry out resolution unless a third Int (cross-core) is also present; the cross-core stimulation of resolution is the result of enhanced cleavage rates by flee partner Ints; Holliday junction resolution involves cleavage at the site of Int binding, i.e., in cis; the accessory proteins and arm-type Int binding sites influence the efficiency and directionality of resolution; the higher-order strictures involved in recombination consist of specific Int bridges mediated by accessory proteins: branch migration is not the principal mechanism of sensing DNA homology during resolution of the all site Holliday junction. 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 are the minimal protein requirements for stimulation of resolution by a cross-core Int? 4) How is the DNA configured in the Holliday junction-protein complex? 5) What dynamic features are important in the formation and resolution of Holliday junctions and what do they depend upon? 6) How do the long-range effects of the att site arms and accessory proteins influence the efficiency and/or directionality of resolution?
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