We are studying control of the cell cycle in the model organism E.coli. Our interests include initiation of replication, chromosome organization and segregation and replication fork collapse and repair.
Chromosome replication is initiated by an initiator protein which binds to the origin, forms an oligomeric complex, separates the two strands of the double helix, and recruits the helicase and the rest of the replication machinery. Initiation must be regulated in such a way that each origin fires once, and only once, per generation. A main actor in proper timing of replication in Escherichia coli is the initiator protein itself, DnaA. DnaA is an AAA+-type protein with structural similarity to archaeal and eukaryotic Orc1/Cdc6 proteins. DnaA is also a transcription factor which interacts with RNA polymerase. The activity of DnaA is regulated by Hda and DnaN (β) and occurs by gradual hydrolysis of active ATP-DnaA to inactive ADP-DnaA as the replisome replicates DNA.
In order to ensure genetic stability, the genetic material must be correctly replicated once per cell cycle and properly segregated to the new daughter cells. Extra rounds of replication are prevented by specific inactivation of new origins by the SeqA protein. This specific inactivation is called origin sequestration and consists for about 1/3 of the cell cycle. SeqA also binds to newly replicated, hemimethylated GATC sites behind the replication forks and is involved in chromosome organization and/ or segregation. The hemimethylated GATC sites are also the basis for directing mismatch repair enzymes to the correct DNA strand.
Errors generated by the replication forks are the cause of many instability mechanisms. A key protein in the replisome is ring-formed and tethers the polymerase to the DNA and makes it processive. This protein is in human cells called PCNA and in bacteria it is called the beta (β) clamp. We are studying several mutations in the beta clamp protein important in preventing replication fork collapse. We will transfer knowledge from the simple E. coli system and generate models for the study of replication fork collapse in human cells.
Although most bacterial genomes are composed of a single chromosome, many unicellular organisms also harbour several chromosomes, giving the possibility to study coordinated replication of several chromosomes in simple systems. Vibrio cholerae, the causative agent of cholera has a large and a small chromosome. Apparently the large chromosome governs the replication of the small chromosome.
Based on knowledge acquired through basic science an applied science project under the EU 5th framework, aimed at development of new types of drugs targeting replication proteins, was established. This resulted in a method (no longer patent protected) for identifying the presence of a protein inhibitor in a sample.