With respect to the HIV Integrase, imaging of the complex will elucidate the structure of the complex formed when HIV-integrase binds the active end of linear DNA which represents the HIV double strand DNA. We used the AFM as the primary imaging tool and have seen the complex form very clearly. We used a range of DNA lengths (90, 150, 350, 500 and 1000 bps) in forming the complexes. The complex formation efficiency depends on the DNA length which implies protein-DNA interactions at DNA sites distal to the active IN binding site. It is interesting that the primary product in these constructs is that two DNA molecules bind to a protein tetramer and these complexes go on to aggregate by binding among the protein oligomers, forming spider-like structures. We quantified the volumes of the bound particles in two-DNA/protein complexes and confirmed them to be consistent with bound tetramers. We also examined the integration process to """"""""host"""""""" DNA and observe stable synaptic complexes as well as fully integrated DNA. IN also appears to affect the conformational state of DNA molecules away from their binding ends, as we observe supecoil-like structures formed by two distinct such linear molecules. We are in the process of clarifying this phenomenon which would shed light into the detailed mechanics of integration complex nuclear transport and would explain the dependence of IN binding affinity to DNA length even though IN appears to bind DNA only at its active ends. In addition we are forming integration products with """"""""host"""""""" DNA to study the topology of half-site integration products, the first step in the actual viral DNA insertion into the host DNA. Analyzing large numbers of images of the complex may also give insights into the topological arrangement of the DNA relative to the protein thus clarifying the structure-function of IN further. ? ? With respect to the VDJ gene fragment processing by RAG1 and RAG2 proteins we form the complex under different physiological conditions and use the AFM for imaging. Proteins RAG1 and RAG2 bind the recombination signal sequences (RSS) on the VDJ gene segments and cause a double strand break before recombination can take place. Imaging the complex we established the stoichiometry of the bound protein complex and we are in the process of clarifying the topological arrangement of the DNA relative to the protein complex. In addition, each both RAG1 and RAG2 contain a maltose binding domain (MBP) and we are using specific antibodies to these domains to further clarify the arrangement of the constituent components of the functional complex.
