Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/138691
Title: SINGLE MOLECULE STUDIES OF DNA-H-NS INTERACTIONS IN PROKARYOTIC CELLS
Authors: RANJIT S GULVADY
Keywords: H-NS, DNA, magnetic tweezers, nucleation site
Issue Date: 27-Jul-2017
Citation: RANJIT S GULVADY (2017-07-27). SINGLE MOLECULE STUDIES OF DNA-H-NS INTERACTIONS IN PROKARYOTIC CELLS. ScholarBank@NUS Repository.
Abstract: Bacterial DNA is assembled within the cell with the help of a class of proteins called the Nucleoid Associated Proteins (NAPs). The Histone-like Nucleoid Structuring Protein, or H-NS, has been shown to be one of the major NAPs that assists in the silencing of potentially virulent genes. While a lot of knowledge on H-NS has been obtained via biochemical studies, the molecular mechanisms behind its gene silencing function are not as well studied. For my PhD, I performed biophysical studies on the H-NS protein using the magnetic tweezers. To begin with, DNA sequence-dependent H-NS binding was investigated. H-NS is known to bind to specific high-affinity AT-rich nucleation sites with the aid of its C-terminus. The propensity of H-NS to bind to one such site - the 10 bp proU nucleation site - was demonstrated by performing a DNA sequence-dependent study. Next, in order to gain a better understanding of the mechanism employed by H-NS in DNA binding, the role of the H-NS linker that connects the C and N-terminal was investigated. Point mutations on the linker resulted in a sharp decrease in the binding affinity in comparison to wt H-NS, suggesting a charge-dependent mechanism for DNA-binding, and highlighting the importance of the H-NS linker in its binding function. In our final study, H-NS-DNA binding experiments were done with a longer DNA, where two proU nucleation sites were placed apart from one another. These experiments suggested that H-NS initially identifies the two nucleation sites before undergoing local spreading to completely cover the DNA. Taken together, these results strengthen our understanding of H-NS-DNA binding, and offer a potential mechanism by which H-NS carries out its function of gene silencing.
URI: http://scholarbank.nus.edu.sg/handle/10635/138691
Appears in Collections:Ph.D Theses (Open)

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