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Bacterial DNA Replication

Prof. Dr. Ute Curth
Medizinische Hochschule Hannover
Biophysical Chemistry
OE 4350

Tel.:

+49-511-532-9372

Fax.:

+49-511-532-5966


e-mail: (Ute Curth)

 

General Information

The highly accurate duplication of the complete genetic material is essential for the nearly error-free transmission of genetic information to the next generation. This highly complex process is carried out by several proteins including DNA-polymerases, RNA-polymerases, exonucleases, helicases and single-stranded DNA binding proteins.

Schematic representation of the E. coli replication fork, emphasizing the protein-protein interactions at the lagging strand.

In the bacterial cell the main enzyme complex responsible for DNA replication is the DNA polymerase III holoenzyme. This holoenzyme is composed of 10 different subunits, which can be divided into three distinct subcomplexes. The core polymerase contains the α-subunit, which represents the actual polymerase activity, the β-sliding clamp tethers the holoenzyme to its DNA substrate and is responsible for processive DNA synthesis and the clamp-loader complex is necessary for the opening of the ring-shaped sliding clamp to load it onto the DNA template. Each DNA polymerase holoenzyme contains two core polymerases, which are held together by the τ-subunits of the clamp loader complex (see Figure).

Owing to the 5'-3' direction of DNA synthesis and the antiparallel nature of double-stranded DNA, both DNA strands have to be synthesized in opposite directions. Therefore, only the leading strand can be synthesized continuously, whereas the lagging strand has to be synthesized in so called Okazaki fragments, comprising approximately 1000 nucleotides. Each of these Okazaki fragments has to be started by the action of the primase, which provides the DNA polymerase with RNA primers.

Due to the delayed action of the lagging strand polymerase, stretches of single-stranded DNA (ssDNA) occur at the lagging strand, which have to be protected against nucleolytic attack. This task is fulfilled by the single-stranded DNA binding (SSB) protein, which is also capable of preventing DNA hairpin formation and therefore configures the ssDNA for the action of the polymerase. The SSB protein interacts physically both with the primase and the clamp loader complex, an interaction, which is enhanced in the presence of ssDNA. The interaction of the SSB/ssDNA complex with primase prevents the premature dissociation of the RNA primer and the competing interaction with the clamp loader displaces the primase and allows the loading of the β-sliding clamp onto the template/primer complex. The interactions of SSB are mediated by its highly-conserved C-terminal region, which is essential for the survival of the bacterial cell. Protein-protein interactions of this C-terminus also play a crucial role in replication restart and in DNA repair.

Our main interest lies in the protein-protein and protein-DNA interactions occurring at the bacterial replication fork and the structural investigation of protein complexes. We use E. coli as a host for expression of recombinant proteins and protein complexes. To date, we purified several components of the E. coli DNA replication system and started the characterization of their in vitro properties. Currently we are extending our investigation to the replication systems of other bacterial organisms like Pseudomonas aeruginosa.

Group

Left to Right:
Lidia Litz, Technical Assistant
Andrea Bogutzki M. Sc.
Ute Curth
, Prof. Dr. rer. nat (Group Leader)
Natalie Naue, Dr. rer. nat.

Publications

  • Krausze, J., Probst, C., Curth, U., Reichelt, J., Saha, S., Schafflick, D., Heinz, D.W., Mendel, R.R., and Kruse, T. (2017).
    Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor.
    Biochem J 474, 163-178.

  • Zumbragel, F.K., Machtens, D.A., Curth, U., Luder, C.G., Reubold, T.F., and Eschenburg, S. (2017).
    Survivin does not influence the anti-apoptotic action of XIAP on caspase-9.
    Biochem Biophys Res Commun 482, 530-535.

  • Vamosi, G., Mucke, N., Muller, G., Krieger, J.W., Curth, U., Langowski, J., and Toth, K. (2016).
    EGFP oligomers as natural fluorescence and hydrodynamic standards.
    Sci Rep 6, 33022.

  • Reubold, T. F., K. Faelber, N. Plattner, Y. Posor, K. Ketel, U. Curth, J. Schlegel, R. Anand, D. J. Manstein, F. Noe, V. Haucke, O. Daumke and S. Eschenburg (2015).
    Crystal structure of the dynamin tetramer.
    Nature 525(7569): 404-408.

  • Lee, C., Wigren, E., Trcek, J., Peters, V., Kim, J., Hasni, M.S., Nimtz, M., Lindqvist, Y., Park, C., Curth, U., Lunsdorf, H., and Romling, U. (2015).
    A novel protein quality control mechanism contributes to heat shock resistance of worldwide-distributed Pseudomonas aeruginosa clone C strains.
    Environ Microbiol 17, 4511-4526.

  • Zhao, H., R. Ghirlando, C. Alfonso, F. Arisaka, I. Attali, D. L. Bain, M. M. Bakhtina, D. F. Becker, G. J. Bedwell, A. Bekdemir, T. M. Besong, C. Birck, C. A. Brautigam, W. Brennerman, O. Byron, A. Bzowska, J. B. Chaires, C. T. Chaton, H. Colfen, K. D. Connaghan, K. A. Crowley, U. Curth, T. Daviter, W. L. Dean, A. I. Diez, C. Ebel, D. M. Eckert, L. E. Eisele, E. Eisenstein, P. England, C. Escalante, J. A. Fagan, R. Fairman, R. M. Finn, W. Fischle, J. G. de la Torre, J. Gor, H. Gustafsson, D. Hall, S. E. Harding, J. G. Cifre, A. B. Herr, E. E. Howell, R. S. Isaac, S. C. Jao, D. Jose, S. J. Kim, B. Kokona, J. A. Kornblatt, D. Kosek, E. Krayukhina, D. Krzizike, E. A. Kusznir, H. Kwon, A. Larson, T. M. Laue, A. Le Roy, A. P. Leech, H. Lilie, K. Luger, J. R. Luque-Ortega, J. Ma, C. A. May, E. L. Maynard, A. Modrak-Wojcik, Y. F. Mok, N. Mucke, L. Nagel-Steger, G. J. Narlikar, M. Noda, A. Nourse, T. Obsil, C. K. Park, J. K. Park, P. D. Pawelek, E. E. Perdue, S. J. Perkins, M. A. Perugini, C. L. Peterson, M. G. Peverelli, G. Piszczek, G. Prag, P. E. Prevelige, B. D. Raynal, L. Rezabkova, K. Richter, A. E. Ringel, R. Rosenberg, A. J. Rowe, A. C. Rufer, D. J. Scott, J. G. Seravalli, A. S. Solovyova, R. Song, D. Staunton, C. Stoddard, K. Stott, H. M. Strauss, W. W. Streicher, J. P. Sumida, S. G. Swygert, R. H. Szczepanowski, I. Tessmer, R. T. t. Toth, A. Tripathy, S. Uchiyama, S. F. Uebel, S. Unzai, A. V. Gruber, P. H. von Hippel, C. Wandrey, S. H. Wang, S. E. Weitzel, B. Wielgus-Kutrowska, C. Wolberger, M. Wolff, E. Wright, Y. S. Wu, J. M. Wubben and P. Schuck (2015).
    A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.
    PLoS One 10(5): e0126420.

  • Ghirlando, R., H. Zhao, A. Balbo, G. Piszczek, U. Curth, C. A. Brautigam and P. Schuck (2014). Measurement of the temperature of the resting rotor in analytical ultracentrifugation.
    Anal Biochem 458: 37-39.

  • Kuhle, K., J. Krausze, U. Curth, M. Rossle, K. Heuner, C. Lang and A. Flieger (2014).
    Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity.
    J Biol Chem 289(27): 18657-18666.

  • Naue, N., M. Beerbaum, A. Bogutzki, P. Schmieder and U. Curth (2013)
    The helicase-binding domain of Escherichia coli DnaG primase interacts with the highly conserved C-terminal region of single-stranded DNA-binding protein.
    Nucleic  Acids Res.  41(8): 4507-4517

  • Zhao, H., R. Ghirlando, G. Piszczek, U. Curth, C. A. Brautigam and P. Schuck (2013)
    Recorded Scan Times Can Limit the Accuracy of Sedimentation Coefficients in Analytical Ultracentrifugation.
    Anal  Biochem. 437(1): 104-108

  • Ringel, P., J. Krausze, J. van den Heuvel, U. Curth, A. J. Pierik, S. Herzog, R. R. Mendel and T. Kruse (2013).
    Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.
    J Biol Chem 288(20): 14657-14671.

  • Lakshminarasimhan, M., U. Curth, S. Moniot, S. Mosalaganti, S. Raunser and C. Steegborn (2013). Molecular architecture of the human protein deacetylase Sirt1 and its regulation by AROS and resveratrol.
    Biosci Rep 33(3).

  • Naue, N. and U. Curth (2012)
    Investigation of protein-protein interactions of single-stranded DNA-binding proteins by analytical ultracentrifugation.
    Methods Mol. Biol. 922: 133-149.

  • Wyszomirski, K. H., U. Curth, J. Alves, P. Mackeldanz, E. Moncke-Buchner, M. Schutkowski, D. H. Kruger and M. Reuter (2012)
    Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.
    Nucleic Acids Res. 40(8): 3610-3622

  • Fonfara I., Curth U., Pingoud A., Wende W. (2012).
    Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases.
    Nucleic Acids Res. 40(2):847-860

  • El Houry Mignan S., Witte G., Naue N., Curth U. (2011).
    Characterization of the chi psi subcomplex of Pseudomonas aeruginosa DNA polymerase III.
    BMC Mol Biol. 12(1):43.

  • Linkner, J., Witte, G., Stradal, T., Curth, U. and Faix, J. (2011)
    High-Resolution X-Ray Structure of the Trimeric Scar/WAVE-Complex Precursor Brk1
    PLoS One 6, e21327

  • Winkler, I., A. D. Marx, D. Lariviere, R. Heinze, M. Cristovao, A. Reumer, U. Curth, T. K. Sixma and P. Friedhoff  (2011)
    Chemical trapping of the dynamic MutS-MutL complex formed in DNA mismatch repair in Escherichia coli
    J Biol Chem 286, 17326-17337

  • Breitsprecher, D., A. K. Kiesewetter, J. Linkner, M. Vinzenz, T. E. Stradal, J. V. Small, U. Curth, R. B. Dickinson and J. Faix (2011)
    Molecular mechanism of Ena/VASP-mediated actin-filament elongation.
    Embo J. 30: 456-467.

  • Naue, N., R. Fedorov, A. Pich, D. J. Manstein and U. Curth (2011)
    Site-directed mutagenesis of the χ subunit of DNA polymerase III and single-stranded DNA-binding protein of E. coli reveals key residues for their interaction.
    Nucleic Acids Res. 39(4): 1398-407

  • Hoffmann, A., P. N. Dannhauser, S. Groos, L. Hinrichsen, U. Curth and E. J. Ungewickell (2010)
    A comparison of GFP-tagged clathrin light chains with fluorochromated light chains in vivo and in vitro
    Traffic 11(9): 1129-40

  • Arad G., Hendel, A., Urbanke, C., Curth, U. and Livneh, Z. (2008)
    Single-stranded DNA-binding protein recruits DNA polymerase V to primer termini on RecA-coated DNA
    J. Biol. Chem, 283 (13), 8274-8282

  • Witte, G., Fedorov, R. and Curth, U. (2008)
    Biophysical analysis of Thermus aquaticus single-stranded DNA binding protein
    Biophys. J., 94 (6), 2269-79

  • Curth, U. und Urbanke, C. (2007)
    Analytische Ultrazentrifugation: Charakterisierung von Protein-Protein Wechselwirkungen.
    BIOspektrum 13 (6), 643-645

  • Fedorov, R., Witte, G., Urbanke, C., Manstein, D.J. and Curth, U. (2006)
    3D structure of Thermus aquaticus single-stranded DNA-binding protein gives insight into the functioning of SSB proteins.
    Nucleic Acids Res, 34, 6708-6717.

  • Urbanke, C., Witte, G. and Curth, U. (2005)
    Sedimentation velocity method in the analytical ultracentrifuge for the study of protein-protein interactions.
    Methods Mol Biol, 305, 101-114

  • Witte, G., C. Urbanke and U. Curth (2005)
    Single-stranded DNA-binding protein of Deinococcus radiodurans: a biophysical characterization.
    Nucl. Acids Res.  33 (5): 1662-1670.

  • Witte, G., C. Urbanke and U. Curth (2003)
    DNA polymerase III chi subunit ties single-stranded DNA binding protein to the bacterial replication machinery.
    Nucl. Acids. Res. 31(15): 4434-4440

  • Landwehr, M., U. Curth and C. Urbanke (2002)
    A dimeric mutant of the homotetrameric single-stranded DNA binding protein from Escherichia coli.
    Biol Chem 383(9): 1325-33

  • Genschel, J., Curth, U., Urbanke, C. (2000)
    Interaction of E. coli single-stranded DNA binding protein (SSB) with exonuclease I. The carboxy-terminus of SSB is the recognition site for the nuclease
    Biological Chemistry 381, 183-192

  • Carlini,L., Curth,U., Kindler,B., Urbanke,C., Porter,R.D. (1998)
    Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli
    FEBS Letters 430, 197-200

  • Yang, C., Curth, U., Urbanke, C. and Kang, C.-H. (1997)
    Crystal structure of human mitochondrial single-stranded DNA binding protein at 2.4Å resolution
    Nature structural biology  4, 153

  • Webster, G., Genschel, J., Curth, U. Urbanke, C., Kang, C. and Hilgenfeld, R. (1997)
    A common core for binding single-stranded DNA: Structural comparison of the single-stranded DNA-binding proteins from E. coli and human mitochondria
    FEBS Letters  411, 313-316.

  • Genschel, J., Litz, L., Thole, H., Roemling, U. and Urbanke, C. (1996)
    Isolation, sequencing and overproduction of the single-stranded DNA binding protein from Pseudomonas aeruginosa PAO
    Gene 182, 137-143

  • Curth, U., Genschel, J., Urbanke, C. and Greipel, J. (1996)
    In vitro and in vivo function of the carboxyterminus of E. coli single-stranded DNA binding protein
    Nucleic Acids Research 24, 2706-2711

  • Misselwitz, R., Welfle, K., Curth, U., Urbanke, C. and Welfle, H. (1995)
    Stability of Escherichia coli single-stranded DNA binding protein (EcoSSB)
    Journal of Biomolecular Structure and Dynamics  12, 1041-1054

  • Curth, U., Urbanke, C., Greipel, J., Gerberding, H., Tiranti, V. and Zeviani, M. (1994)
    Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties
    European Journal of Biochemistry 221, 435-443


  • Carlini, L.E., Porter, R.D., Curth, U. and Urbanke, C. (1993)
    Viability and preliminary in vivo characterization of site-directed mutants of Escherichia coli single-stranded DNA-binding protein
    Mol.Microbiol. 10, 1067-1075

  • Curth, U., Greipel, J., Urbanke, C. and Maass, G. (1993)
    Multiple binding modes of the single-stranded DNA binding protein from Escherichia coli as detected by tryptophan fluorescence and site-directed mutagenesis
    Biochemistry 32, 2585-2591

  • Curth, U., Bayer, I., Greipel, J., Mayer, F., Urbanke, C. and Maass, G. (1991)
    Amino Acid 55 plays a central role in tetramerization and function of E. coli single stranded DNA binding protein
    Eur.J.Biochem. 196, 87-93