Doctor of Philosophy (PhD)
Ku is central to the non-homologous end-joining pathway of DNA double strand break repair, first discovered in eukaryotes and more recently in prokaryotes and archaea. This study concerns the importance of two unique sequence features of Ku protein from Mycobacterium smegmatis that include a lysine-rich extension at the C-terminus and a zinc-binding motif in the DNA-binding bridge-region. The unique C-terminal tail of M. smegmatis Ku contains several lysine-rich low-complexity PAKKA repeats that are absent from homologs encoded by obligate parasitic mycobacteria, but present in other mycobacterial proteins such as histone-like proteins. Removal of the lysine-rich extension from Ku decreased thermal stability and abolished DNA end-joining. The tail contacts the core DNA binding domain of Ku and hinders DNA-protein interaction as evidenced by an increase in DNA binding affinity upon removal of the lysine-rich extension. In contrast to Ku lacking the C-terminus, full-length Ku can directly bind DNA without free ends and form multiple complexes with a short stem-loop-containing DNA previously designed to accommodate only one Ku dimer, suggesting that these properties are conferred by its C-terminus. My study suggests that low-complexity lysine-rich sequences have evolved repeatedly to modulate the function of unrelated DNA-binding proteins and that extensions beyond the shared core domain may have independently evolved to expand Ku function. An in vitro metal binding assay showed zinc binding to a predicted zinc-binding motif in the bridge-region of M. smegmatis Ku, an event that stabilizes the protein and prevents cysteine oxidation, but has little effect on DNA binding. In vivo, zinc induced significant upregulation of the gene encoding Ku as well as a divergently oriented gene encoding a predicted zinc-dependent MarR family transcription factor. In addition, overexpression of Ku conferred zinc tolerance on E. coli. I speculate that zinc binding sites in Ku proteins from M. smegmatis and other mycobacterial species have been evolutionarily retained to provide protection against zinc toxicity. In all, my study identifies novel properties conferred by unique sequences present in M. smegmatis Ku protein, which suggests that the retention and evolution of unique sequences within a protein provides an adaptive advantage to microorganisms against environmental stress.
Document Availability at the Time of Submission
Student has submitted appropriate documentation to restrict access to LSU for 365 days after which the document will be released for worldwide access.
Kushwaha, Ambuj Kumar, "Functional significance of unique sequences in Mycobaterium smegmatis Ku proteins" (2013). LSU Doctoral Dissertations. 1359.