Bacterial DNA Replication
Prof. Dr. Ute Curth
Division of Structural Biochemistry, OE8830
Hannover Medical School
Phone: +49 511 532 9372
Fax: +49 511 532 5966
The highly accurate duplication of the complete genetic material is a prerequisite for the division of the bacterial cell. This highly complex process is carried out by several proteins and protein complexes including DNA polymerases, RNA polymerases, exonucleases, helicases and single-stranded DNA-binding proteins (SSB). 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 fragments of about 1000 nucleotides, the so-called Okazaki fragments. Since DNA polymerases are not able to start DNA synthesis de novo, each Okazaki fragment has to begin with an RNA primer provided by primase.
In the bacterial cell the main enzyme complex responsible for DNA replication is the DNA polymerase III (pol III) holoenzyme that consists of three sub-complexes: The core polymerase, the β-sliding clamp and the clamp-loader complex. Each pol III holoenzyme contains two core polymerases held together by the τ-subunits of the clamp loader complex (see Figure).
Stretches of single-stranded DNA that occur at the lagging strand are protected by SSB against nucleolytic attack and hairpin formation. SSB also interacts both with the primase and the χ subunit of the clamp loader complex via its highly-conserved C-terminal region that is essential for the survival of the bacterial cell.
Our main interest lies in the processes occurring at the lagging strand of the bacterial replication fork that enable the switch between primase and polymerase activity. Therefore, we investigate the respective protein-protein and protein-DNA interactions and examine the structures of the protein complexes. To this end, we purified the components of the E. coli DNA replication system and established several in vitro DNA replication assays.