Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The sirtuin protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modifications. Here, we explored the effect of lysine acetylation on E. coli topoisomerase I through analysis of TopA relaxation activity and protein expression in cell extract of wild-type and a ΔcobB mutant strains. We showed that the absence of deacetylase CobB in a ΔcobB mutant reduced intracellular TopA relaxation activity while elevating TopA expression and topA gene transcripts levels. Acetyl phosphate mediated lysine acetylation decreased the activity of purified TopA in vitro, and the interaction with purified CobB protected TopA from such inactivation. We explored the physiological significance of TopA acetylation on DNA supercoiling by two-dimensional gel analysis and on cell growth rate by growth curve analysis. We found that the absence of CobB increased negative DNA supercoiling. The slow growth phenotype of the ∆cobB mutant can be partially compensated by overexpression of recombinant TopA. In addition, the specific activity of TopA expressed from His-tagged fusion construct in the chromosome was inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. Investigation of TopA relaxation mechanism using nuclease footprinting and TopA oxidative crosslinking suggested the potential association of TopA acetylation in catalysis. Mass spectrometry analysis of in vitro acetyl phosphate acetylated TopA identified abundant lysine acetylation sites. Substitution of lysine residues by site-directed mutagenesis was used to model the effect of acetylation on individual lysine residues. Our results showed that substitution of Lys-484 with alanine reduced the relaxation activity, suggesting the reduction of TopA relaxation activity by acetylation was probably in part due to acetylation on Lys-484. These findings demonstrate that E. coli topoisomerase I is modulated by lysine acetylation and the prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the sirtuin deacetylase CobB.
| Identifer | oai:union.ndltd.org:fiu.edu/oai:digitalcommons.fiu.edu:etd-4345 |
| Date | 28 June 2017 |
| Creators | Zhou, Qingxuan |
| Publisher | FIU Digital Commons |
| Source Sets | Florida International University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | FIU Electronic Theses and Dissertations |
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