Prof. Dr. Ute Curth
Medizinische Hochschule Hannover
e-mail: (Ute Curth)
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.
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.