Study of the dynamics of higher order protein DNA complexes involved in variety of DNA transactions

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Dynamic aspects of the chromosomes that are the target of transposing viruses such as bacteriophage Mu and HIV are studied in this project. A pair of DNA cleavages and strand transfers involving the ends of Mu or HIV DNA sequence and their interaction with chromosomal target DNA sites take place within a context of higher order protein-DNA assemblies for each DNA segments involved. The transposing viral DNA ends are assembled into higher order protein-DNA complexes called transpososome (for Mu) or preintegration complex (for HIV), the core of which is composed of two end segments of the transposing viral DNA synapsed by a tetramer of MuA transposase or HIV IN protein. The assembly of these higher order protein-DNA complexes in nature takes place in the presence of additional DNA binding proteins that influence the assembly process. The target DNA for transposition also exists as higher order protein-DNA complexes in the form of eukaryotic chromosomes or bacterial nucleoid. How the transposing viral DNA complex assembly process and the activity of the chromosomal DNA as the target of transposition are influenced by general chromosome/nucleoid associated proteins, which impact the dynamic behavior of DNA is not well understood. In both prokaryotes and eukaryotes, chromosomal DNA molecules are kept in dynamic condensed states involving large variety of DNA condensation proteins. These proteins either bend/flex DNA, or cross-bridge distant DNA segments through direct, or indirect DNA binding. In our separate project (DK036165), we also study the mechanism of bacterial chromosome/plasmid partition systems that involve the ParA/B/S class of system components. ParA and ParB proteins also cause DNA condensation in highly controlled fashion, and their functional dynamics most likely are impacted by other DNA condensation proteins associated with bacterial nucleoid, NAPs (nucleoid associated proteins). This project aims to advance our understanding of how the chromosome-associated proteins, through their influence on condensed DNA dynamics, affect DNA transactions such as transposing viral DNA integration, transposition target site search, as well as bacterial chromosome segregation processes. HIV DNA within a preintegration complex is protected by BAF protein from self-destructive auto-integration. BAF is believed to condense DNA in a way that makes it inaccessible for self-destructive auto-integration. The mechanism of DNA condensation by BAF was studied at a single DNA-molecule level by using fluorescence labeled BAF and a high-sensitivity fluorescence microscope system. BAF is a small dimeric protein that binds two DNA segments in near orthogonal orientations, forming cross-bridges to condense DNA. BAF is thought to play roles after mitosis during re-assembly of nucleus. While it is not considered to be a general chromosome-associated condensation protein, it serves as a model bridging-type DNA condenser, and we study its DNA interaction dynamics along with bacterial NAPs and other DNA condensing proteins. Experimental approaches are currently under development to quantitatively evaluate the impacts of DNA-condensing proteins, from both prokaryotic and eukaryotic origins, on the structural dynamics of DNA, on higher order protein-DNA complex assembly process, and the dynamic properties of these large molecular assemblies.