Title page for ETD etd-11082004-113624


Type of Document Dissertation
Author Ghosh, Sharmistha
Author's Email Address sghosh1@lsu.edu
URN etd-11082004-113624
Title DNA Binding Properties of Histone-Like Protein HU from Deinoccus radiodurans Suggest Involvement in DNA Recombination
Degree Doctor of Philosophy (Ph.D.)
Department Biochemistry (Biological Sciences)
Advisory Committee
Advisor Name Title
Anne Grove Committee Chair
David Donze Committee Member
John R. Battista Committee Member
Patrick J. Dimario Committee Member
Daniel B. Paulsen Dean's Representative
Keywords
  • bacterial chromatin
  • dna bending
  • type ii dna binding protein
  • holliday junction
  • dna flexibility
Date of Defense 2004-10-22
Availability unrestricted
Abstract
The Histone-like protein HU is ubiquitous in eubacteria. Usually with a length of ~90 amino acids, they are predominantly homodimeric, with sequence and structural homology. Escherichia coli HU is involved in DNA repair and recombination. The crystal structure of Anabaena HU shows that it binds DNA with prolines intercalating into the DNA backbone, introducing two kinks at a spacing of 9 bp and bending the DNA through a variable angle of 105-140. Deinococcus radiodurans is a gram positive mesophile, capable of reconstituting its genome from 1000-2000 double strand breaks incurred due to exposure to environmental extremes.

In the first study, D. radiodurans HU (DrHU) is characterized in terms of its DNA binding properties. The binding site size of DrHU is the largest so far reported, ~50 bp. DrHU binds preferentially to four-way junction DNA with half-maximal saturation of 18 2 nM. In distinct contrast to E. coli HU, DrHU has no marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able of mediating circularization of linear duplex DNA.

In the second study, the N-terminus of DrHU was truncated, generating ΔDrHU, and the functional role of the N-terminus investigated. ΔDrHU exhibits a binding site size of 17 1 bp similar to HU homologs from other mesophiles. ΔDrHU also binds preferentially to four-way junction DNA, but protects the crossover rather than the junction arms protected by DrHU. The melting temperature of ΔDrHU of 46.4 0.1C is similar to that of HU from mesophiles. DrHU interacts with other D. radiodurans proteins(s) in the presence of four-way junction DNA, suggesting its role in DNA recombination.

In a similar study with the HU homolog from Helicobacter pylori (HpyHU), the protein binds stably to four-way junction DNA with half-maximal saturation of 5.0 0.5 nM. Thermal denaturation of HpyHU measured by circular dichroism spectroscopy yields a Tm = 56.4 0.1C suggesting greater than average thermal stability. Mutagenesis of HpyHU suggests that a differential target site selection of HU proteins is achieved through their individual capacity for inducing the required DNA bend.

Files
  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  Ghosh_dis.pdf 3.22 Mb 00:14:53 00:07:39 00:06:41 00:03:20 00:00:17

Browse All Available ETDs by ( Author | Department )

If you have questions or technical problems, please Contact LSU-ETD Support.