The use of synthetic Holliday junctions and covalent modifications to investigate the mechanisms of strand exchange and higher-order complex assembly in lambda site -specific recombination

This volume begins with a review of the lambda Int family of topoisomerases and recombinases---while special attention is focused on aspects of the eponymous lambda Int and its site specific recombination system, the discussion is set against recent discoveries in related systems that illuminate our understanding of lambda. The third chapter describes experiments performed with synthetic Holliday junctions that challenge a long-standing model of strand exchange that invoked 7 bp of branch migration and replace it with a model that employs pairwise swaps of 2--3 nt DNA segments. In the fourth chapter this model is expanded to incorporate insights gained from the solution structure of the Holliday junction to show that top- and bottom-strand exchanges are globally symmetrical---the strands that are poised to swap are more sharply bent at the branch point than the opposite pair in both reactions. The fifth and sixth chapters describe the use of DNA-based biotin dT modifications to investigate the mode of binding by Int to core and arm sites: lambda Int is shown to bind both faces of the core-type DNA helix with the CB and catalytic domains located on opposite sides, and the behavior of Int with arm-site modifications supports a theoretical model of interaction between the N-terminal domain of Int and the P'1 arm site. In chapter seven, large synthetic Holliday junctions that mimic the intermediate of integrative recombination and harbor biotin dT modifications at individual arm sites plus pairs of mutant sequences at the core (to selectively disrupt binding by the N- and C-terminal domains of Int respectively) are used to infer the existence of bridging interactions during resolution by lambda Int. Each of the arm sites in the P' arm (P'1, P'2, and P '3) is shown to make a partial contribution to core delivery and resolution recovery. Possible models are discussed in the final chapter.