Research Overview

Fig 1: Human HeLa cells were exposed to 5 gray of gamma irradiation. After 2 h, cells were fixed and the localizations of gammaH2AX (red, DNA repair foci) and human replication protein A subunit p32 (green) were determined. DNA was stained with DAPI (blue). Merge: combination of the red and green fluorescence.

In eukaryotes chromosomal DNA replication is a highly coordinated and tightly controlled process. To this end the replication proteins ORC (origin recognition complex), CDC6, CDT1, MCM2 to MCM7 (minichromosome maintenance), CDC45, DNA polymerase alpha-primase, and RPA (replication protein A) must co-operate during the eukaryotic cell cycle.

The activity of the replication and regulatory proteins is controlled by posttranslational modifications and protein-protein interactions. In order to analyze these physical interactions, we have used various biochemical and cell biological techniques such as; co-immunoprecipitation, fluorescence (cross) correlation spectroscopy, a modified ELISA technique, fluorescence recovery after photobleaching (FRAP), functional test systems, and biophysical methods.

Moreover, we have produced phosphorylation mutants of DNA polymerase alpha-primase and RPA as well as phosphopeptide-specific antibodies to investigate the functions of these proteins in the cell cycle and after DNA damage. To study their activities in living cells we expressed fusion proteins of GFP and wild type as well as mutant proteins in human cells and have carried fluorescence correlation spectroscopy and FRAP.

The human polyoma virus BKV is often activated after kidney transplantations and can then cause polyomavirus-associated nephropathy. To study the life cycle of the virus we have established a cell-free BKV DNA replication system.