Konstruktion synthetischer sekundärer Chromosomen zur Charakterisierung von DNA-Reparatur und Segregation in Escherichia coli

All functions of each cell are encoded in the genome, which is distributed equally to the daughter cells during each cell division. This applies to chromosomes, regardless the number of chromosomes. The integrity of the genome is essential for the survival of any organism. Using synthetic biology methods, extensive alterations of genomes or entire chromosomes can be synthesized, although the focus is mostly on the coding sequences. However, chromosomes are more than merely a sequential arrangement of genes. Chromosomes need systems for replication, segregation, organization, and repair, which is often done by interactions of proteins with DNA sequence motifs. The present study investigates such so-called chromosome maintenance systems using synthetic secondary chromosomes in E. coli. Can the results generate a better understanding of DNA replication in bacteria? The synthetic secondary chromosome (synVicII) established in this work replicates in E. coli similarly to the secondary chromosome in V. cholerae on which it is based. After an initial characterization, the design of synVicII was further optimized. A crucial step was to generate compatibility of synVicII with the hierarchical DNA assembly system MoClo. In parallel, an approach was established to generate highly variable, long DNA sequences in which user-defined DNA motifs can be excluded. The insertion of these DNA sequences into synVicII allowed the construction of synthetic secondary chromosomes with a size of 100 kb. These chromosomes make it feasible for the first time to analyze the interaction of DNA segregation and DNA mismatch repair in vivo by a set of three synthetic secondary chromosomes. Both processes are dependent on the presence of hemi-methylated GATC sequences in E. coli. This work shows that a differentiated binding of the two responsible proteins, for the above processes, SeqA and MutH, can be achieved by an ordered GATC arrangement to allow comparative analysis. Since the functionality of SeqA has not yet been fully understood, the quantitative understanding of SeqA was experimentally improved. Based on this data a model for SeqA structure variants at the replication fork was generated. It was demonstrated by FRAP experiments that SeqA is a dynamic protein that diffuses freely between two binding events within the cell. SeqA and Dam compete for the hemi-methylated GATCs. It was shown that both proteins are in a similar ratio to each other. This could be an aspect for how GATC re-methylation is regulated. The results of the present work contribute significantly to the understanding of DNA replication in bacteria.