RECOMBINATION INTERMEDIATES -- PROPERTIES & PROCESSING

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This proposal is an extension of ongoing studies on the formation, properties and resolution of the DNA recombination intermediate (four-way DNA junction) known as a Holliday junction (HJ). The model system used here, the Int-dependent site-specific recombination pathway of bacteriophage lambda, has been extensively characterized at both the genetic and biochemical levels. It belongs to a large family of proteins from archaebacteria, eubacteria, and yeast that catalyze rearrangements between DNA sequences with minimal homology to each other. Recombinases from this family have the unique capacity to both generate and resolve HJs without the input of energy. Holliday junctions are an important intermediate in many biological pathways that rearrange DNA and an understanding of their properties should contribute to the body of basic knowledge that serves as a platform for health-related advances in the area of genetics. The proposed experiments grow out of recent biochemical, genetic, and structural data that afford insights into how HJs are generated and resolved by the Int protein, which is also a paradigm for the large subset of virally-encoded family members that are hetero-bivalent DNA binding proteins. The particular insights which drive this proposal concern the mechanisms of communication between Int protomers during the strictly ordered sequence of DNA cleavages, ligations and isomerizations and the architecture of a ternary complex containing Int, HJ, and "arm-type" DNA sites. The experiments can be grouped into four general questions: a) What determines the precise order of strand exchanges during formation of the HJ intermediate? b) During HJ resolution, what is the mechanism that ensures only one of the two partner Int pairs will be "on stage" (active) at one time? c) How do arm-type DNA sites increase the fidelity of HJ resolution? d) What does the ternary HJInt- arm-type DNA complex look like at high resolution?