DNA replication in Escherichia coli: a comprehensive study of the Tus-Ter complex
Moreau, Morgane Julie Jackie (2013) DNA replication in Escherichia coli: a comprehensive study of the Tus-Ter complex. PhD thesis, James Cook University.
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Abstract
The circular chromosome of bacteria is replicated by two replisomes assembled at the unique origin and moving in opposite direction until they meet at specific termination sites. The process of DNA replication termination is the stage of replication that is the least understood, both in prokaryotes and eukaryotes. In E. coli, the termination protein Tus binds to 14 termination sites (TerA-J, TerK, L, TerY, Z) spread throughout the genome. The intriguing organization and symmetry of Ter sites has puzzled scientists for decades. The Tus-Ter complex is polar and blocks replication forks approaching from one direction but not from the other. Most Ter sites are oriented to form a fork trap so that convergent forks can enter and merge in the terminus region but not exit. However, the significance of having maintained such a wide fork trap remains unclear. The mechanism responsible for the polarity of the Tus-Ter complex is still being debated. A protein-protein interaction between the DnaB helicase at the forefront of the replisome and Tus bound to Ter has been proposed (Bastia et al., 2008, Mulugu et al., 2001). The alternative mechanism involves the formation of the Tus-Ter-lock (TT-lock) where Tus captures the cytosine at position 6 in the Ter core sequence upon duplex unwinding by DnaB and becomes locked on Ter thereby preventing DnaB translocation (Mulcair et al., 2006). Since the discovery of the TT-lock, there has been no further investigation on its formation in the remaining Tus-Ter complexes. However, the proportion of fork pausing at each Ter sites has previously been determined in vivo and was detected at seven Ter sites (TerA-D, TerG, TerH and TerI). The remaining Ter sites were classified as pseudo-Ter (Duggin and Bell, 2009). Nevertheless, all Ter were able to arrest forks in an artificial context, yet with varying efficiencies (Duggin and Bell, 2009). This prompted the question of whether or not the outer Ter sites maintained their biological function. This work provides the first comparative study of the ten primary Ter sites (TerA-J) in terms of their affinity and specificity for Tus and whether they are all able to form a TT-lock. The variation in affinity and TT-lock forming ability of Ter sites was compared to their intrinsic efficiency in arresting a replisome and to the in vivo distribution of Tus on Ter sites. Finally, ectopic Ter sites were inserted into the E. coli genome to determine the effect of TT-lock formation on cell growth. Several new methods were developed during this thesis for the characterization of Tus-Ter and Tus-Terlock complexes in a time and cost-effective manner.
The Ter sites were shown to be different both in terms of their affinity for Tus and in their ability to form a TT-lock. Six strong Tus binding sites (TerA-E and TerG) were identified and the outermost TerH, TerI and TerJ were classified as moderate binders. The binding of Tus to TerF was only marginally stronger than a non-specific DNA region of the oriC. The strong binders were all able to form a strong TT-lock whereas moderate binders varied in their TTlock forming efficiencies. TerF and TerH were unable to form significant locks. The affinity and TT-lock forming efficiencies of the Ter sites correlated well with their intrinsic pausing efficiency determined by Duggin and Bell (2009). In the cell, Tus was distributed onto Ter sites according to their intrinsic affinity. It was demonstrated that only the strong Ter sites are able to cause significant fork arrest suggesting that replication forks are unlikely to break through the innermost Ter sites and that the outer Ter sites may be used to prevent non-oriC initiated forks to travel towards the origin. A new paradigm is being proposed to explain the multiplicity of Ter sites and the advantage in maintaining such a wide fork trap. Finally, the three new assays developed in this study, GFP-Basta, DSF-GTP and the qPCR-based DNA binding assay, proved to be invaluable tools for the detailed characterization of protein-DNA complexes. These news techniques have considerable applications in both genomic and proteomic programs.
| Item ID: | 31903 |
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| Item Type: | Thesis (PhD) |
| Keywords: | Tus-Ter complex; Tus-Terlock complex; Escherichia coli; E coli; lock formation; characterization of protein-DNA complexes; proteomics; intermolecular interactions |
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| Copyright Information: | Copyright © 2013 Morgane Julie Jackie Moreau |
| Additional Information: | For this thesis, Morgane Moreau received the Dean's Award for Excellence 2014. Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 3. Moreau, Morgane J.J., Morin, Isabelle, and Schaeffer, Patrick M. (2010) Quantitative determination of protein stability and ligand binding using a green fluorescent protein reporter system. Molecular BioSystems, 6 (7). pp. 1285-1292. Chapter 3. Moreau, Morgane J.J, and Schaeffer, Patrick M. (2012) A polyplex qPCR-based binding assay for protein–DNA interactions. Analyst, 137. pp. 4111-4113. Chapter 4. Moreau, Morgane J.J., and Schaeffer, Patrick M. (2012) Differential Tus–Ter binding and lock formation: implications for DNA replication termination in Escherichia coli. Molecular BioSystems, 8 (10). pp. 2783-2791. Chapter 5. Moreau, Morgane J.J., Morin, Isabelle, Askin, Samuel P., Cooper, Alanna, Moreland, Nicole J., Vasudevan, Subhash G., and Schaeffer, Patrick M. (2012) Rapid determination of protein stability and ligand binding by differential scanning fluorimetry of GFP-tagged proteins. RSC Advances, 2 (31). pp. 11892-11900. |
| Date Deposited: | 30 Apr 2014 01:43 |
| FoR Codes: | 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060109 Proteomics and Intermolecular Interactions (excl Medical Proteomics) @ 50% 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060114 Systems Biology @ 50% |
| SEO Codes: | 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50% 97 EXPANDING KNOWLEDGE > 970110 Expanding Knowledge in Technology @ 50% |
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