Functional significance of unique sequences in Mycobaterium smegmatis Ku proteins
type: CreativeWork
description: 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 f…
abstract: 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 f…
author: Kushwaha, Ambuj Kumar
keywords: lysine-rich extension, histone-like proteins, Ku protein, DNA binding, electrophoretic mobility shift assay, low-complexity repeats, non-homologous end-joining, tryptophan fluorescence, thermal stability, zinc-binding, zinc toxicity
date published: 2013-01-01T08:00:00Z
date modified: 2026-07-24T02:59:22Z
provider: Lousiana State University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Biological Sciences

PropertyValue
property ID: DOI
value: 10.31390/gradschool_dissertations.1359