RECOMBINATION INTERMEDIATES: FORMATION, PROPERTIES

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This project is derived from our long term interest in the mechanisms by which genetic material is reassorted and rearranged. We propose to study the formation, properties and resolution of Holliday recombination intermediates. 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. Because the Holliday intermediate figures prominently in so many recombination pathways, the results obtained in the proposed studies should be of general significance -- in addition to their importance for site-specific recombination, and the (growing) Int family of recombinases. The proposed experiments depend upon our recent finding that this pathway proceeds via a sequential highly ordered pair of reciprocal strand exchanges. They also draw on our demonstration that the appropriate "synthetic" recombination intermediates afford a very useful tool for studying Holliday junctions. Six specific questions are addressed in this proposal: 1) What is the role of DNA:DNA homology in initiating strand transfer? 2) What are the role and properties of branch migration in resolution? 3) What are the binding patterns and interactions among the Int proteins bound at the Holliday junction? 4) What is the role of arm-type distal sites in the resolution of Holliday junctions? 5) what are the structural features of the Holliday junction in relation to recombination? 6) Does Int-mediated strand transfer have mechanistic features related to illegitimate recombination?