Replication proteins as new drug targets

1. Research project:
Replication proteins as new drug targets

2. Group:
Kirsten Skarstad (PI), Line Johnsen (postdoc), Solveig Fossum (postdoc), Anne Wahl (engineer), Henning Bråten (undergraduate).

3. Home adress on the net:
http://radium.no/skarstad

4. Institution:
Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital

5. Main aim of research project:
The main aim of the project is to discover new types of antibacterial and anticancer drugs. Knowledge about the mechanisms of action of replication proteins will be used to design screens for drugs that specifically target the replication machinery.

6. Some important recent results:
A conditional mutant of the Escherichia coli initiator protein, DnaA, has been characterized. The mutant grows with normal growth rate at 42°C, but dies at 30°C due to excess initiation (overinitiation). In this strain, there is too much activity of DnaA. Such a strain can be exploited in the search for new antibacterial agents. A potential drug targeting DnaA activity will reduce overinitiation and cause the strain to survive at low temperature. The strain has been improved and made amenable for high-throughput screening by providing an alternative initiation pathway which does not depend on DnaA activity. The strain still shows lethal overinitiation at 30°C, but will not die if DnaA is knocked out. When a potential drug that inhibits DnaA activity, and thus overinitiation, occurs in a screen (detected by recovery of growth at low temperature), it will not be missed if added in a concentration that completely inactivates DnaA. Thus, the new strain (SF53) still dies at 30°C due to overinitiation, but the DnaA independent replication will salvage the strain when DnaA is knocked out. The strain is the basis of an exceptionally robust screen. The phenomenon of lethal overactivity is rarely found in nature and is ideal in a positive screen (K. Skarstad, S. Fossum, W. Messer and C. Weigel (2006) “A screen for novel protein inhibitors”, Patent no EP169115, International Publication no WO 2004/108958 A1).

7. Methods in current use:
Cell cycle analysis by flow cytometry, immunofluorescence and GFP-based localization by microscopy. Strain construction by ET cloning and P1 transduction, plasmid construction by traditional cloning. Identification of genes, transcripts and proteins by PCR, Southern, Northern and Western. Protein purification. Studies of protein-protein interaction, protein-DNA interaction, replication and transcription in vitro.

8. Available equipment:
Institute of Cancer Research Core Facilities: FACS DIVA and LSRII Flow cytometers, DNA sequencing equipment, PhosphorImager, Ultracentrifuge.
Department of Cell Biology: Zeiss Axiovision fluorescence microscope, ChemiGenius gel documentation equipment, PCR machines, scintillation counter, molecular biology- and cell cycle analysis-PC software.

9. Collaborators:

9.1 Heidi Lyng, Department of Radiation Biology, Institute for Cancer Research.
9.2 DnaAcos A/S, Bio-Medisinsk Innovasjon AS, Forskningsparken, Gaustadalleen 21, 0349 Oslo.

9.3 Stefano Donadio (KtedoGen and NAICONS, Milano), Jens von Kries (Leibniz-Institute fur Molekulare Pharmakologie, Berlin), Christoph Weigel (Technical University, Berlin)

10. Is the group interested in joining a larger collaborative project at DNR:
Yes

11. Tentative name of possible collaborative project(s):
Initiation of replication in eukaryotes, prokaryotes and archaea.

12: Some key search words:
DnaA, replication proteins, HT screens