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 donor 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 could influence the assembly process. The target DNA for transposition also exists as higher order protein-DNA complexes in the form of chromosome or 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. This project aims to advance our understanding of how the chromosome-associated proteins, through their influence on condensed DNA dynamics, affect DNA transaction processes such as transposing viral DNA integration, transposition target site search, as well as bacterial chromosome segregation processes. The molecular interactions involved in the Mu transpososome and HIV preintegration complex have been studied by using fluorescence labeled proteins and DNA substrates. This study led to better understanding of the organization of the Mu-end synaptic complex and identification of a new reaction intermediate. HIV DNA within a preintegration complex is protected by BAF protein, which is believed to condense DNA in a way that makes it inaccessible for self-destructive auto-integration. The mechanism of DNA condensation by BAF has been studied at a single DNA-molecule level by using fluorescence labeled BAF and a high-sensitivity fluorescence microscope system. Experimental approaches are currently under development to evaluate the impacts of a variety 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 molecular assemblies.
| Li, Min; Ivanov, Vassili; Mizuuchi, Michiyo et al. (2012) DNA requirements for assembly and stability of HIV-1 intasomes. Protein Sci 21:249-57 |
| Ivanov, Vassili; Li, Min; Mizuuchi, Kiyoshi (2009) Impact of emission anisotropy on fluorescence spectroscopy and FRET distance measurements. Biophys J 97:922-9 |
| Skoko, Dunja; Li, Min; Huang, Ying et al. (2009) Barrier-to-autointegration factor (BAF) condenses DNA by looping. Proc Natl Acad Sci U S A 106:16610-5 |
