| Many proteins bring distantly separated regions of DNA together by binding to two or more sites at a time, forming a synaptic complex. In this dissertation, Atomic Force Microscopy (AFM) was used to characterize three protein systems which require DNA synapsis in order for the enzyme to be active. These include the restriction enzymes EcoRII and SfiI, and the V(D)J recombination activation gene RAG1 and RAG2 (hereafter referred to as RAG1/2). By using AFM imaging, clues about the oligomeric states of the protein, the topology of the DNA, the specificity of DNA binding, and the relative abundance of various intermediate binding states. These clues can then be applied to better understand the intricacies of the mechanism used by the protein to interact with DNA. In addition, by using the newly developed High-Speed Atomic Force Microscopy (HS-AFM) technique, information about sequential events of the binding/unbinding process, as well as information about the translocation of the proteins between DNA sites can be gained by observing the molecules at frame rates hundreds of times faster than previously possible with AFM.;The results of this study identify intricacies of the mechanisms used by each protein. (1) For EcorII, AFM images of the protein interacting with three recognition sites combined with kinetic data from our collaborators on a three-site plasmid identifies this as the first restriction enzyme discovered to have a mechanism requiring more than two sites. Additionally, a high prevalence of intermediate binding events was observed, along with a translocation mechanism which consists primarily of sliding and 3 dimensional diffusion. (2) For SfiI, the observed translocation mechanism differs from the EcoRII results. Dried AFM images show very stable loop formation with translocation consisting primarily of intersegmental transfer events. (3) Lastly, dried images of the RAG1/2 proteins were able to identify a very large volume complex which holds the two recognition sites together. The implications of the findings for each of these proteins will be discussed in more detail throughout the chapters of this dissertation. |
