Lambda site-specific recombination (SSR), like all recombination reactions, requires the synapsis or juxtaposition of two DNA substrates by recombination proteins and their accessory factors into a productive arrangement. The protein-DNA complexes assembled contain several different proteins serving both catalytic and/or architectural roles (e.g., bending DNA). Lambda SSR is now known to comprise four distinct pathways. The integration and excision pathways, used respectively to join and separate the lambda and E. coli genomes, are unidirectional, irreversible reactions where the products differ from the substrates. These two reactions differ in two fundamental ways: they contain different DNA partners (attB and attP vs attL and attR) and they have different protein requirements (int vs int+xis). The two other pathways, between two attL or two attP sites, promoted by Int are bidirectional and the products look like the substrates. Therefore, the four pathways are distinguished both by the structure of the DNA substrates and by the proteins which help Int mediate the reaction: the chemical steps of cleaving and resealing the DNA take place in four distinct protein-DNA environments. This application focuses on examining how protein-DNA and protein-protein interactions in the four pathways determine differences among the pathways, by separating features of Int that are required for chemical catalysis from features that influence directionality. To that end, four specific aims are delineated: 1- A fine-structure analysis of interactions between Int and the regions of strand exchange will be carried out for a second synaptic recombination intermediate to compare these interactions between two bidirectional pathways of recombination. 2- An in-depth in vitro analysis of existing Int variants, which are able to recombine through some pathways but not others, will be performed to determine which features are required for all recombination pathways and which are required for individual pathways. This analysis will determine similarities and differences among the pathways. 3- The nature and role of protein-protein interactions within a recombination complex will be investigated for different pathways. Specifically, the contacts between Int monomers will be determined. 4- A new tool, based on a ligation-defective Int mutant, will be developed to study transient synaptic complexes.
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