Microbiology
Reactions of the DNA translocase FtsK and XerC & XerD recombinases from Pseudomonas aeruginosa
Luhai Xu
Student number 3107041
Supervisor Ian Grainge
Table of Contents
Abstract 3
LIST OF ABBREVIATIONS 4
Chapter1: Introduction 5
Chapter 2 Materials and Methods 26
Chapter 3: Results 41
Chapter 4: Discussion 63
References 74
Appendix 80
Abstract
DNA replication is an important biological process and occurs in all living organisms to copy their DNA. However, chromosome dimers might be formed by homologous recombination between sister chromosome before cell division which is lethal to the cell. Luckily, these chromosome dimers can be resolved back into monomers by a site-specific recombination carried out at the dif site prior to cell divison.
XerC/XerD recombination is an example of site-specific recombination found in almost all bacteria. Three critical factors are involved in this recombination reaction: the DNA translocase FtsK, tyrosine recombinases XerC/XerD and the specific site dif. The chromosome dimer resolution is done after the activation of XerD by the γ sub-domain of FtsK at the dif site. When stimulated by FtsK, XerD exchanges the first pair of strands to form a Holliday junction which is then resolved by XerC.
FtsK is a multidomain protein required for cell division and chromosome partitioning. It consists of an N-terminal domain, linker domain and C-terminal domain. The C-terminal domain is made up of three sub-domains α，β and γ. Its multidomain makes it is multifunctional. FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. It also promotes the XerC/XerD site-specific recombination at dif sites by switching the activity of the XerC/XerD recombinases. XerC and XerD, two recombinases belonging to the lambda integrase family of enzymes, are essential for the Xer site-specific recombination.
This project concerns work on the mechanisms
Luhai Xu
Student number 3107041
Supervisor Ian Grainge
Table of Contents
Abstract 3
LIST OF ABBREVIATIONS 4
Chapter1: Introduction 5
Chapter 2 Materials and Methods 26
Chapter 3: Results 41
Chapter 4: Discussion 63
References 74
Appendix 80
Abstract
DNA replication is an important biological process and occurs in all living organisms to copy their DNA. However, chromosome dimers might be formed by homologous recombination between sister chromosome before cell division which is lethal to the cell. Luckily, these chromosome dimers can be resolved back into monomers by a site-specific recombination carried out at the dif site prior to cell divison.
XerC/XerD recombination is an example of site-specific recombination found in almost all bacteria. Three critical factors are involved in this recombination reaction: the DNA translocase FtsK, tyrosine recombinases XerC/XerD and the specific site dif. The chromosome dimer resolution is done after the activation of XerD by the γ sub-domain of FtsK at the dif site. When stimulated by FtsK, XerD exchanges the first pair of strands to form a Holliday junction which is then resolved by XerC.
FtsK is a multidomain protein required for cell division and chromosome partitioning. It consists of an N-terminal domain, linker domain and C-terminal domain. The C-terminal domain is made up of three sub-domains α，β and γ. Its multidomain makes it is multifunctional. FtsK acts at the bacterial division septum to couple chromosome segregation with cell division. It also promotes the XerC/XerD site-specific recombination at dif sites by switching the activity of the XerC/XerD recombinases. XerC and XerD, two recombinases belonging to the lambda integrase family of enzymes, are essential for the Xer site-specific recombination.
This project concerns work on the mechanisms
References: Afsahi S. (2011). https://confluence.crbs.ucsd.edu/display/CS/FtsZ Aussel L., Barre F Barre F. X., Aroyo M., Colloms S. D., Helfrich A., Cornet F. and Sherratt D. J. (2000). FtsK functions in the processing of a Holliday junction intermediate during bacterial chromosome segregation. Genes Dev. 14, pages 2976-2988 Bartosik, A.A Bebenek K. and Kunkel T. A. (2004). Functions of DNA Polymerases. Advances in Protein Chemistry. Volume 69, pages 137-165 Bennion B Berg J.M., Tymoczko J.L., Stryer L. and Clarke N.D. (2002). Biochemistry. W.H. Freeman and Company. Chapter 27: DNA Replication, Recombination and Repair. Berg J.M., Tymoczko J.L., Stryer L Berg J.M., Tymoczko J.L., Stryer L. and Clarke N.D. (2002). Biochemistry. W.H. Freeman and Company. Chapter 27: Section 4: DNA Replication of Both Strands Proceeds Rapidly from Specific Start Sites Bigot S., Corre J., Louarn J.M., Cornet F., and Barre F.X Bloom K. and Joglekar A. (2010). Towards building a chromosome segregation machine. Nature 463 pages 446-456 Bramhill D Bramhill D. and Kornberg A. (1988). A Model for Initiation at Origins of DNA Replication. Cell, Volume 54, pages 915-918 Cox M K.J. (2000). The importance of repairing stalled replication forks. Nature vol 404 37-41 DePamphilis M Dorazi R., and Dewar S.J. (2000). Membrane topology of the N-terminus of the Escherichia coli FtsK division protein. FEBS Lett 478: pages 13–18 Draper G Dubarry N., Possoz C., and Barre F.X. (2010). Multiple regions along the Escherichia coli FtsK protein are implicated in cell division. Mol Microbiol 78: Pages 1088–1100 Ehrmann M Erfibi and Lutkenhaus J. (1991). FtsZ ring structure associated with division in Escherichia coli. Nature 354, Pages 161 – 164 Erickson H Firshein W. (1989). Complex in prokaryotic DNA replication. Annu. Rev. Microbiol. Volume 43, pages 89-120 Gajiwala K.S., and Burley S.K Geissler B. and Margolin W. (2005). Evidence for functional overlap among multiple bacterial cell division proteins: compensating for the loss of FtsK. Mol Microbiol 58: Pages 596–612 Gordon, G.S Grainge I. (2010). FtsK – a bacterial cell division checkpoint? Molecular Microbiology Volume 78, Issue 5, pages 1055–1057 Grainge I., Lesterlin C Griffiths A. J. F., Wessler S. R., Lewontin R. C. and Carroll S. B. (2008). Introduction to Genetic Analysis. W.H. Freeman and COmpany. Chapter 7: DNA: Structure and Replication. pages 283-290 Hiasa H Hengen P. N. (1995). Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences. Volume 20, Issue 7, pages 285-286 Jakimowicz D, Chater K, Zakrzewska- Czerwinska J Jensen R. B. and Shapiro L. (1999). Chromosome segregation during the prokaryotic cell division cycle. Current Opinion in Cell Biology, Volume 11, Issue 6, Pages 726-731 Jemsen J Kees L. M. C., Franken, Hoebert S., Hiemstra, Krista E. Meijgaarden V., Yanri S., Lemon K.P. and Grossman A.D. (2000). Movement of replicating DNA through a stationary replisome. Mol Cell.; 6: 1321–30 Kornberg (1984) Lee J. Y., Finkelstein L. J., Estelle C. Sherratt D. J. and Greene E. C. (2010) Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK PNAS Volume 109 no. 17 pages 6531-6536 Lemon K.P Lewin C.S. and Amyes S.G.B. (1991). The role of the SOS response in bacteria exposed to zidovudine or trimethoprim. J Med Microbiol vol. 34 no. 6 329-332 Life Science News 18 (2004) Liu G., Draper G. C. and Donachie W. D. (2002). FtsK is a bifunctional protein involved in cell division and chromosome localization in Escherichia coli. Molecular Microbiology Volume 29, Issue 3, pages 893–903 Lutkenhaus J Marians K. J. (1992). Prokaryotic DNA Replicaion. Annual Review of Biochemistry. Volume 61 pages 673-715 Margolin W Massey T.H., Mercogliano C.P., Yates J., Sherratt D.J., and Lowe J. (2006). Double-stranded DNA translocation: structure and mechanism of hexameric FtsK. Mol Cell 23: pages 457–469 McCulloch S.D, and Kunkel T.A Pease P. J., Levy O., Cost G. J., Gore J., Ptacin J. L., Sherratt D., et al. (2005). Sequence-directed DNA translocation by purified FtsK. Science 307: pages 586–590 Recchia G Sciochetti S. A., Piggot P. J. and Blakely G. W. (2001). Identification and characterization of the dif site from Bacillus sutilis. Journal Bacteriol 183, pages 1058-1068 Sinden R Sinden R. R. (1994). DNA structure and function. page 52 Sivanathan V., Allen M.D., de Bekker C., Baker R., Arciszewska L.K., Freund S.M Sophie N., Pages C., Rousseau P., Bourgeois P. L. and Cornet F. (2010). Are two better than one? Analysis of an FtsK/Xer recombination system that uses a single recombinase. Nucl. Acids Res. 38 (19):pages 6477-6489 Spiers A Steiner W., Liu G., Donachie W. D. and Kuempel P. (1999). The cytoplasmic domain of FtsK protein is required for resolution of chromosome dimers. Molecular Microbiology Volume 31, Issue 2, pages 579–583 Stenlund A., (2003) Subramanya H. S. , Arciszewska L. K., Baker R. A., Bird L. E., Sherratt D. J. and Wigley D. B. (1997). Crystal structure of the site-specific recombinase, XerD. The EMBO JOURNAL 16, PAGES 5178-5187 Weigel C., Schmidt A., Ryckert B., Lurz R Weiss D. S. (2004). Bacterial cell division and the septal ring. Molecular Microbiology Volume 54, Issue 3, pages 588–597 Yates J., Aroyo M., Sherratt D.J