Synthetic secondary chromosomes in E. coli
To learn more about the biology of bacterial chromosome maintenance we establish a synthetic secondary chromosome in Escherichia coli as experimental system. Chromosome maintenance mechanisms usually consist of a DNA binding protein and the respective DNA sequence motif. The distribution of such DNA motifs on the chromosome often determines the function of the system. On a synthetic secondary chromosome the motif distribution can be according to any design. By construction and characterization of synthetic chromosome variants we want to understand more about the role of DNA motif distribution in chromosome biology. The template for our synthetic secondary chromosome is Vibrio cholerae. This bacterium carries a natural secondary chromosome. We have constructed a 10kb “mini-chromosome” based on the replication origin of the V. cholerae secondary chromosome. The mini-chromosome replicates in E. coli and presents the chassis for more synthetic secondary chromosomes to come.
DNA replication of the two Vibrio cholerae chromosomes
The genome of eukaryotic organisms is divided on multiple chromosomes while bacteria usually have only one chromosome. An interesting exception is Vibrio cholerae, the causative agent of the cholerae disease. This bacterium carries a secondary chromosome (about 1 Mbps) in addition to the primary chromosome (3 Mbps). For three reasons this two-chromosome system came into focus of scientists over the last years. First, targeting the unique replication of the secondary chromosome could open up possible routes to therapeutics development specific for Vibrio species. Second, Vibrio is used as template for synthetic secondary chromosomes in heterologous hosts. Third, the two chromosomes in V. cholerae present the simplest possible system to study general questions of multi-chromosome systems. For all these aspects a deep understanding of the V. cholerae chromosome biology is essential. We study the DNA replication of Vibrio cholerae chromosomes using a wide range of molecular biology and bioinformatics approaches.
The role of SeqA in E. coli DNA replication and chromosome segregation
SeqA is a fascinating protein with the unique ability to bind highly specific to hemi-methylated GATC sites. Such hemi-methylation is characteristic for newly replicated DNA. While the old DNA strand is methylated the new strand will be un-methylated. This situation lasts until the Dam methyltransferase adds the methyl-group at the N6 position of the adenine. Binding of SeqA to hemi-methylated GATCs at the replication origin hinders immediate re-initiation which is critical to guarantee proper timing of replication rounds. In addition SeqA follows the newly replicated DNA behind the replication fork potentially in a treadmilling-like fashion, with a SeqA structure growing at the leading end and shrinking in the trailing end. The moving of SeqA behind the replication fork must be based on the combination of DNA-polymerase progression, re-methylation by Dam and SeqA binding and leaving the DNA. We want to uncover the details of this molecular treadmilling mechanism using a combination of molecular biology, simulation and bioinformatics.