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Research

Group Leader: Dr. Heinz-Peter Nasheuer
Phone No.: 353-91-49 24 30
Fax No.: 353-91-49 55 04
Email: heinz.nasheuer

nuigalway.ie
Postal Address: National University of Ireland, Galway, Dept. of Biochemistry, University Road, Galway, Ireland.

Group Members:

Heinrich Anhold (postdoctoral researcher, jointly with Prof. Noel Lowndes)
Ronan Broderick (postgrad. student)
Aoife Corduff (postgrad. student)
Ronan Flemming (postgrad. student)
Delia Lowery (postgrad. student)
Cathal Mahon (postdoctoral researcher)
Maren Oehlmann (postdoctoral researcher)
Kristina Rehmet (postgrad. student)
Holger Stephan (postdoctoral researcher)

Regulation and Mechanisms of DNA Replication

Control of cell division is a fundamental process in all organisms. Cells have developed a regulatory network to control the cell cycle, to initiate DNA replication at the right time and to prevent untimely DNA replication.

The mechanisms that control the onset of S-phase most likely have their target in proteins involved in the initiation of DNA replication but the linkage of these processes is still not fully understood. We investigate the linkage between the cell cycle and DNA replication. Using a combination of cell biological and biochemical approaches, we study the general principles that determine how the initiation of DNA replication is controlled by the cell cycle regulatory network. Specific aims are to study the regulation of the activity of Cdc45, DNA polymerase alpha-primase, the eukaryotic, single-stranded DNA-binding protein RPA, Poly-(ADP ribose)-polymerase, and the tumour supressor gene product p53 by cell cycle dependent protein kinases. As model systems we study DNA replication in cycling human cells and viral DNA replication in vitro by purified proteins.

Replication of Papovavirus DNA

Viruses were first described as disease-causing agents that can multiply only in cells. Since bacteriophages, plant and animal viruses are greatly dependent on their hosts, they have been extensively used to study cellular functions. Although the papovaviruses use similar strategies to replicate in their hosts, mouse polyomavirus (mPyV) multiplies only in mouse cells, and simian virus 40 (SV40) propagates in primate cells. Biochemical studies of SV40 and mPyV DNA replication revealed that the host specificity could be reproduced in vitro: Mouse cell extracts support replication of mPyV DNA in vitro , while human cell extracts exclusively replicate SV40 DNA.

The cellular DNA polymerase alpha-primase is the species-specific factor in both viral DNA replication systems. We cloned the cDNAs that encode for all four subunits of mouse and human polymerase alpha-primase and expressed them in insect cells. The initiation of mPyV DNA replication in vitro is a species-specific reaction and mouse p48 subunit is primarily responsible for the species-specificity of mPyV DNA replication in vitro. In contrast to these results, DNA polymerase alpha-primase, that contains the human p180, synthesizes oligoribonucleotides in SV40 initiation and replicates SV40 DNA in depleted mouse extracts. We conclude that the human p180 subunit determines host specific replication of SV40 DNA in vitro. Interestingly, the enzyme complex HHMM (contains human p180 and p68, together with mouse p58 and p48) was highly active in both viral replication systems. We are currently investigating the mechanisms controlling DNA replication of human polyomavirus BK.

Financial Support

Health Research Board, Ireland
Irish Research Council for Science, Engineering and Technology (IRCSET)
National Institute of Health (NIH), USA
Science Foundation Ireland

Publication List

Research Publications

48. Taneja, P., Nasheuer, H.P., Hartmann, H., Grosse, F., Fanning, E. und Weisshart, K. (2007). Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication. Biochem J., 407: 313-320.

47. Taneja, P., Boche, I., Hartmann, H., Nasheuer, H.P., Grosse, F., Fanning, E. und Weisshart, K. (2007). Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication. FEBS Lett., 581: 3973-3978.

46. Pollok, S., Bauerschmidt, C., Sänger, J., Nasheuer, H.P. und Grosse, F. (2007) Human Cdc45 is a proliferation-associated antigen FEBS J. 274: 3669-3684.

45. Bauerschmidt, C., Pollok, S., Kremmer, E., Nasheuer, H.P.* und Grosse F.* (2007) Interactions of Human Cdc45 with the Mcm2-7 complex, the GINS complex and DNA polymerases delta and epsilon during S phase. Genes to Cells 12: 745-358. *correspondence.

44. Braet, C., Stephan, H., Dobbie, I. M., Togashi, D. M., Ryder, A. G., Foldes-Papp, Z., Lowndes N. und Nasheuer, H. P. (2007). Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy. Exp Mol Pathol. 82: 156-162.

43. Rytkonen, A. K., Hillukkala, T., Vaara, M., Sokka, M., Jokela, M., Sormunen, R., Nasheuer, H. P., Nethanel, T., Kaufmann, G., Pospiech H. and Syväoja, J. E. (2006). DNA polymerase epsilon associates with the elongating form of RNA polymerase II and nascent transcripts. FEBS J. 273: 5535-5549.

42. Liu, P., Barkley, L.R., Day, T., Bi, X., Slater, D.M., Alexandrow, M.G., Nasheuer, H.P., und Vaziri C. (2006) The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25a/Cdk2-independent mechanism. J Biol Chem, 281: 30631-30644.

41. Rytkönen, A. K., Vaara, M., Nethanel, T., Kaufmann, G., Sormunen, R., Läärä, E., Nasheuer, H. P., Rahmeh, A., Lee, M. Y.W.T., Syväoja, J. und Pospiech, H. (2006) Distinctive activities of DNA polymerases lpha, delta and psilon during human DNA replication. FEBS J. 273: 2984-3001.

40. Beyer, A., Hollunder, J., Nasheuer, H. P. and Wilhelm, T. (2004). Posttranscriptional expression regulation in the yeast Saccharomyces cerevisiae on a genomic scale. Mol. Cell. Proteomics. 3: 1083-1092

39. Weisshart, K., Friedl, S., Taneja, P., Nasheuer, H. P., Schlott, B., Grosse, F. and Fanning, E. (2004). Partial proteolysis of SV40 T antigen reveals intramolecular contacts between domains and conformation changes upon hexamer assembly. J. Biol. Chem. 279: 38943-38951

38. Weisshart, K., Pestryakov, P., Smith, R. W., Hartmann, H., Kremmer, E., Lavrik, O. and Nasheuer, H. P. (2004). Coordinated regulation of replication protein A activities by its subunits p14 and p32 . J. Biol. Chem. 279: 35368-35376.

37. Wilhelm, T., Nasheuer, H.P. und Huang, S. (2003) Physical and functional modularity of the protein network in yeast. Mol. Cell. Proteomics. 2: 292-298

36. Pestryakov, P.E., Weisshart, K., Schlott, B., Khodyreva, S. N., Kremmer, E., Grosse, F., Lavrik, O.I., and Nasheuer, H. P. (2003) Human Replication Protein A: The C-terminal RPA70 and the central RPA32 domains are involved in the interactions with the 3'-end of a primer-template DNA. J. Biol. Chem., 278: 11515-115224

35. Smith R.W.P. und Nasheuer, H.P. (2003) Initiation of JC Virus DNA replication in vitro by human and mouse DNA polymerase alpha-primase. Eur. J. Biochem. 270: 2030-2037

34. Pollok, S., Stoepel, J., Bauerschmidt, C., Kremmer, E., and Nasheuer, H. P . (2003) Regulation of eukaryotic DNA replication at the initiation step by Cdc45 and DNA polymerase a phosphorylation. Biochemical Society Transactions, 31 : 266-269

33. Smith, R.W.P. and Nasheuer, H. P. (2002) Control of complex formation of DNA polymerase alpha-primase and cell-free DNA replication by the C-terminal amino acids of the largest subunit p180. FEBS Letters, 527: 143-146

32. Smith, R.W.P., Steffen, C., Grosse, F., and Nasheuer, H. P. (2002) Species specificity of Simian Virus 40 DNA replication in vitro requires multiple functions of human DNA polymerase alpha. J. Biol. Chem., 277: 20541-20548

31. Melle, C. and Nasheuer, H. P. (2002) Physical and functional interactions of the tumor suppressor protein p53 and DNA polymerase alpha-primase. Nucl. Acids Res. 30: 1493-1499

30. Schub, O., Rohaly, G., Smith, R.W.P., Schneider, A., Dehde, S., Dornreiter, I., and Nasheuer, H. P. (2001) Two independent phosphorylation sites of cellular DNA polymerase alpha-primase cooperate to regulate initiation of simian virus 40 DNA replication. J. Biol. Chem., 276: 38076-38083

29. Kautz, A. R., Weisshart, K., Schneider, A., Grosse, F., and Nasheuer, H. P. (2001) Aminoacids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro. J. Virol. 75: 8569-8578

28. Søe, K., Dianov, G., Nasheuer, H. P., Bohr, V. A., Grosse, F., and Stevnsner, T. (2001) A human topoisomerase I cleavage complex is recognized by an additional human topoisomerase I molecule in vitro. Nucl. Acids Res. 29: 3195-3203

27. Dehde, S., Rohaly, G., Schub, O., Nasheuer, H. P., Bohn, W. Chemnitz, J., Deppert, W., and Dornreiter, I. (2001) Two immunologically distinct human DNA polymerase alpha-primase subpopulations are involved in cellular DNA replication. Mol. Cell. Biol. 21: 2581-2593

26. Kautz, A. R., Schneider, A., Weisshart, K., Geiger, C., and Nasheuer, H. P. (2001) Different regions of primase subunit p48 control mouse polyomavirus and simian virus 40 DNA replication in vitro. J. Virol. 75: 1751-1760

25. Weisshart, K., Förster, H., Kremmer, E., Schlott, B., Grosse, F., and Nasheuer, H. P. (2000) Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system. J. Biol. Chem., 275: 17328-17337

24. Kolpashchikov, D. M., Nasheuer, H. P., Weisshart, K., Ivanova, T. M., Bogachev, V. S., Favre, A., and Lavrik, O. I. (2000) Synthesis of base-substituted dUTP analogs carrying photoreactive 2-Nitro-5-azidobenzoyl group attached to 5'-position of uridine and its application to study human replication protein A by photoaffinity labeling . Bioconjug. Chem., 11: 445-451

23. Lavrik O.I., Kolpashchikov D.M., Weisshart K., Nasheuer H. P. , Khodyreva S. N., and Favre A. (1999) Alternative RPA conformations monitoring by the size of 5'-protruding template strand of DNA duplex. Nucl. Acids Res. 27: 4235-4240.

22. Kolpashchikov, D. M., Weisshart, K., Nasheuer, H. P., Khodyreva, S. N., Fanning, E., Favre, A., and Lavrik, O. I. (1999) Interaction of the RPA p70 subunit with a DNA template directs the p32 subunit to the 3´-end of nascent DNA. FEBS Lett., 450: 131-134.

21. Kühn, C., Müller, F., Melle, C., Nasheuer, H. P., Janus, F., Deppert, W., and Grosse, F. (1999). Surface plasmon resonance measurements reveal stable complex formation between tumorsuppressor gene product p53 and DNA polymerase alpha-primase. Oncogene, 18: 769-774

20. Voitenleitner, C., Rehfuess, C., Hilmes, M., O´Rear, L., Liao, P. C., Gage, D. A., Ott, R., Nasheuer, H. P., and Fanning, E. (1999). Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation . Mol. Cell. Biol., 19: 646-656

19. Lavrik, O.I., Kolpashchikov, D. M., Nasheuer, H. P., Weisshart, K., and Favre, A. (1998). Alternative RPA conformations are detected by crosslinks with primers carrying photoreactive group at 3'-end. FEBS Lett., 441 : 186-190

18. Schneider, A., Smith, R. W. P., Kautz, A. R., Weisshart, K., Grosse, F. and Nasheuer, H. P. (1998). Primase activity of the DNA polymerase alpha-primase complex: Divalent cations stabilize the enzyme activity of the p48 subunit. J. Biol. Chem. 273: 21608-21615.

17. Dantzer, F., Nasheuer, H. P., Vonesch, J.-L., de Murcia, G., and Ménissier-de Murcia, J. (1998). Poly (ADP-ribose) polymerase links the DNA damage surveillance network to the DNA replication machinery. Nucl. Acids Res. 26: 1891-1898.

16. Lavrik, O. I., Nasheuer, H. P., Weisshart, K., Wold, M. S., Prasad, R., Beard, W. A., Wilson, A. H., and Favre, A. (1998). Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases. Nucl. Acids Res. 26: 602-607.,

15. Nesper, J., Smith, R. W. P., Kautz, A. R., Sock, E., Wegner, M., Grummt, F., and Nasheuer, H. P. (1997). A cell-free replication system for human polyomavirus JC DNA. J. Virol. 71: 7421-7428.

14. Voitenleitner, C., Fanning, E., and Nasheuer, H. P. (1997). Phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases regulates initiation of DNA replication in vitro . Oncogene 14: 1611-1615.

13. Stadlbauer, F., Voitenleitner, C., Brückner, A., Fanning, E., and Nasheuer, H. P. (1996). Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase. Mol. Cell. Biol. 16: 94-104.

12. Brückner, A., Stadlbauer, F., Guarino, L. A., Brunahl, A., Schneider, C., Rehfuess, C., Prives, C., Fanning, E., and Nasheuer, H. P. (1995). The mouse DNA polymerase alpha-primase subunit p48 mediates species- specific replication of polyomavirus DNA in vitro. Mol. Cell. Biol. 15: 1716-1724.

11. Stadlbauer, F., Brueckner, A., Rehfuess, C., Eckerskorn, C., Lottspeich, F., Förster, V., Tseng, B. Y. and Nasheuer, H. P. (1994). DNA replication in vitro by recombinant DNA-polymerase- alpha-primase. Eur. J. Biochem. 222: 781-793.

10. Nasheuer, H. P., von Winkler, D., Schneider, C., Dornreiter, I., Gilbert, I., and Fanning, E. (1992). Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system. Chromosoma 102: S52-S59.

9. Pearson, B. E., Nasheuer, H. P., and Wang, T. S. (1991). Human DNA polymerase a gene: sequences controlling expression in cycling and serum-stimulated cells. Mol. Cell. Biol. 11: 2081-2095.

8. Nasheuer, H. P., Moore, A., Wahl, A. F., and Wang, T. S. (1991). Cell cycle-dependent phosphorylation of human DNA polymerase alpha. J. Biol. Chem. 266: 7893-7903.

7. Podust, V. N., Lavrik, O. I., Nasheuer, H. P., and Grosse, F. (1989). DNA polymerase alpha-DNA primase from human placenta. Immunoaffinity purification and preliminary characterization . FEBS Lett. 245: 14-16.

6. Bialek, G., Nasheuer, H. P., Goetz, H., and Grosse, F. (1989). Exonucleolytic proof-reading increases the accuracy of DNA synthesis by human lymphocyte DNA polymerase alpha-DNA primase . EMBO J. 8: 1833-1839.

5. Bialek, G., Nasheuer, H. P., Goetz, H., Behnke, B., and Grosse, F. (1988). DNA polymerase alpha-DNA primase from human lymphoblasts . Biochim. Biophys. Acta 951: 290-297.

4. Nasheuer, H. P. and Grosse, F. (1988), DNA polymerase alpha-primase from calf thymus: Determination of the polypeptide responsible for primase activity. J. Biol. Chem. 263: 8981-8988

3. Nasheuer, H. P. and Grosse, F. (1987). Immunoaffinity-purified DNA polymerase alpha displays novel properties. Biochemistry 26: 8458-8466.

2. Grosse, F., Nasheuer, H. P., Scholtissek, S. and Schomburg, U. (1986). Lactate dehydrogenase and glyceraldehyde-phosphate dehydrogenase are single-stranded DNA-binding proteins that affect the DNA-polymerase-alpha-primase complex . Eur. J. Biochem. 160 : 459-467.

1. Freimüller-Kreutzer, B., Nasheuer, H. P., and Müller, W. H. (1985). Induction of alkaline phosphatase by retinoic acid. Biol. Chem. Hoppe Seyler 366: 317-321.

         Reviews

10. Oehlmann, M., Mahon, C., and Nasheuer, H. P. (2007). Comparison of DNA replication in Xenopus laevis and Simian Virus 40. Adv Exp Med Biol 604: 3-16.
9.      Nasheuer, H. P., Pospiech, H., and Syväoja, J. (2007) Progress towards the anatomy of the eukaryotic DNA replication fork In: Lankenau DH (ed) Genome Integrity: Facets and Perspectives, vol 1. Springer, Berlin Heidelberg DOI: 10.1007/7050_012.
8.      Nasheuer, H. P ., Pospiech, H., and Syväoja, J. (2004) Human DNA Polymerases In : (D. Ganten and K. Ruckpaul eds.) Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine, Springer, Berlin, in the press
7.      Nasheuer, H. P ., Smith, R. W. P., Bauerschmidt, C., Grosse, F., and Weisshart, K. (2002) Initiation of Eukaryotic DNA Replication - Regulation and Mechanisms. Prog. Nucl. Acid Res. Mol. Biol., 72 : 41-94
6.      Bauerschmidt, C., Kremmer, E., and Nasheuer, H. P . (2001) Initiation of Eukaryotic DNA-Replication - A Protein Network Is Decoded. Bioforum, 24 : 589-592
5.       Smith, R. W. P. and Nasheuer, H. P . (2000) DNA replication systems in vivo and in vitro as tools to study virus host interactions . Seite 67-92, In : "Recent Research Developments in Virology" Pandalai (ed.), Transworld Research Network, Trivandrum, Indien,
4.       Hübscher, U., Nasheuer, H. P., and Syväoja, J. (2000) Eukaryotic DNA poly merases, a growing family . Trends Biochem. Sci., 25 : 143-147.
3.       Schneider, C., Winkler, D. v., Dornreiter, I., Nasheuer, H. P. , Gilbert, I., Dehde, S., Arthur, A., and Fanning, E. (1992), Bovine RP-A functions in SV40 DNA replication in vitro, but bovine DNA polymerase < -primase inhibits replication completely 385-397; In: Regulatory Mechanisms of DNA Replication (M. Kohiyama & P. Hughes, eds.); Springer Verlag, Berlin, Heidelberg, New York.
2.       Hartmann, G. R., Biebricher, C., Glaser, S. J., Grosse, F., Katzameyer, M. J., Lindner, A. J., Mosig, H., Nasheuer, H. P. , Rothman-Denes, L. B., Schäffner, A. R., Schnider, G. J., Stetter, K.-O., and Thomm, M. (1988). Twelfth Fritz Lipmann lecture. Initiation of transcription--a general tool for affinity labeling of RNA polymerases by autocatalysis . Biol. Chem. Hoppe Seyler 369 : 775-788.
1.       Grosse, F. and Nasheuer, H. P. (1988), Structure and properties of the im munoaffinity-purified DNA polymerase a -primase complex , 397-402, In: Cancer Cells Vol. 6, Eukaryotic DNA Replication; (B. Stillman & T. Kelly, eds.); Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

Biochemistry, School of Natural Science.
National University of Ireland, Galway, University Road, Galway, Ireland.
Phone: +353 (0)91 492420
E-mail: Biochemistry
This page was last updated Saturday, December 01, 2007