DNA Binding Properties of Histone-Like Protein HU from Deinoccus radiodurans Suggest Involvement in DNA Recombination
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 ?DrHU, and the functional role of the N-terminus investigated. ?DrHU exhibits a binding site size of 17 ± 1 bp similar to HU homologs from other mesophiles. ?DrHU also binds preferentially to four-way junction DNA, but protects the crossover rather than the junction arms protected by DrHU. The melting temperature of ?DrHU of 46.4 ± 0.1°C 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 ± 0.1°C 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.
Advisor:David Donze; John R. Battista; Patrick J. Dimario; Daniel B. Paulsen; Anne Grove
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Keywords:biochemistry biological sciences
Date of Publication:11/11/2004