Single-DNA Studies of Architectural Proteins involved in Bacterial Pathogenesis and Meiosis in Saccharomyces Cerevisiae

Abstract

Both eukaryotic and prokaryotic cells must keep their chromosomal DNA well organized. Packaging of millimeter-long DNA molecules inside bacterial cells and centimeter-to-meter-long ones inside eukaryotic cells is achieved through a number of DNA binding architectural proteins. In bacteria, chromosomal DNA is packaged into a tightly folded nucleoid structure by about a dozen nucleoid-associated proteins (NAPs). In eukaryotic cells, DNA is organized into chromatin by histone proteins. Besides packaging DNA, architectural proteins also play other roles in various critical cellular processes, such as gene transcription regulation and cell division. In the preparation of this thesis, I investigated the gene regulation functions of H-NS, a major NAP in Gram-negative bacteria, which controls pathogenesis of Salmonella, Escherichia coli (E.coli) and Yersinia. My studies revealed the mechanism by which H-NS mediated gene-silencing can be relieved through interaction with another protein, SsrB. I also investigated the DNA-binding properties of MDP1 and mIHF, two acid-fast Gram-positive bacteria proteins expressed in Mycobacterium tuberculosis. These proteins are known to control bacterial growth and regulate entry into the dormant state, but the molecular mechanisms were poorly understood. I found that both of these proteins condense DNA into a stable structure. This suggests they function to protect DNA against reactive oxygen intermediate by host immune system and thus play a role in bacterial growth regulation. Finally, I studied the DNA-binding behavior of the protein Hop1, which plays a critical role in aligning two sister chromatids during meiosis in the eukaryote Saccharomyces cerevisiae. I found that Hop1 mediates tight DNA bridging in a zinc ion dependent manner, which has important physiological implications. All these studies were based on direct measurement using a combination of single-DNA manipulation and atomic force imaging technologies to address fundamental questions concerning the mechanical aspects of interactions between architectural proteins and single-DNA molecules.