Question

Describe the results you would get back if you performed a Chip-Seq experiment for an E. coli strain containing plasmid RK2, to investigate where DnaA-ATP binding occurs throughout the genome. Will you find sequences from multiple regions of the genome?

Answer 1

DnaA protein is both a initiation of replication and transcription factor, which binds to the conserved sequences called DnaA boxes present in the origin of replication. Other than OriC, the datA sites contains multiple DnaA boxes, which regulates the concentration of DnaA inside the cell thereby, preventing incomplete replication initiation and in timing the replication initiation relative to the cell growth.Therefore, there are multiple DnaA binding regions in the genome. In addition the plasmid RK2 also contains DnaA boxes in its origin of replication.

Now coming to Chromatin immunoprecipitation-Sequencing experiment, it is used to identify the DNA binding regions distributed in the genome of a particular DNA binding protein. Following binding of the DNA binding protein to its target site on the genome, other segments of the DNA are degraded by the addition of exonuclease. Then, an antibody specific to the DNA binding protein is added to precipitate the bound regions from the unbound region, hence why it is called as immunoprecipitation. The DNA segments were then extracted and sequenced from the DNA - protein - antibody complex.

Therefore at the end of Chip-Seq experiment for an E.coli strain containing plasmid RK2 you will find sequences from multiple regions such as OriC, DARS and datA of the genome were the DnaA - ATP binding occurs.