Doctor of Philosophy (PhD)
Eubacteria encode numerous small basic histone-like proteins (such as HU, H-NS and Fis) that are required for nucleoid organization and for regulation of DNA-dependent processes. One of these histone-like proteins, HU from Escherichia coli has been shown to associate with the nucleoid and to regulate processes such as DNA repair and recombination. In contrast, the divergent HU homologs encoded by mycobacteria have been variously identified as involved in the physiology of dormancy, in the response to cold shock or as laminin binding proteins associated with the cell envelope. Using indirect fluorescent antibody microscopy, contrary to previous reports, it is shown that the HU-related histone-like protein Hlp from Mycobacterium smegmatis is nucleoid-associated. No evidence of surface exposed Hlp was found in cells treated for cell wall permeabilization. Quantitative Western blots indicate that exponentially growing cells contain ~120 molecules per cell, with up-regulation of Hlp after cold shock estimated to be ~10 fold. Hlp binds both DNA and RNA in vitro and protects DNA from hydroxyl radical- or DNase I-mediated damage. Hlp, which in addition to the HU fold, has a basic C-terminal tail composed of PAAK and PAKK repeats, has extremely high affinity for DNA. The binding affinity of Hlp for 76 bp linear DNA is greater, Kd = 0.037 Â± 0.001 nM, compared to Hlp lacking the C-terminal repeats, Kd = 2.5 Â± 0.05 nM and the C-terminal repeat domain, Kd = 0.82 Â± 0.17 nM. Hlp lacking the entire C-terminal domain does not bind DNA up to 0.5 ÂµM protein concentration. Hlp does not constrain DNA supercoil in the presence of Topoisomerase I but enhances DNA end-joining in the presence of T4 DNA ligase, and this property is mediated by the C-terminal repeats. At <100 nM concentration, Hlp represses transcription by T7 RNA polymerase in vitro whereas the individual N- and C-terminal domains do not, even when added together. These data indicate Hlp domains contribute to high-affinity DNA binding. Combined, the data suggest that its primary functional role may be the DNA dependent responses to environmental stress rather than nucleoid organization.
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Mukherjee, Anirban, "Histone-like protein from Mycobacterium smegmatis has two DNA binding domains and is localized to the nucleoid in vivo" (2007). LSU Doctoral Dissertations. 3779.