Bacterial chromosomes are circular and have a single origin of replication. The repeated sequences in these chromosomes indicate that leading and lagging strand synthesis start at the origin and then continue bi-directionally until they meet, ~180o away at the dif site, a recombination site that separates linked circles. This is in direct conflict with the replication fork trap model that has been posited in the field of DNA replication for the last 30 years. A series of polar termination sites are thought to catch the replication forks between them. This would lead to a gradient of leading and lagging strand signal and a smooth transition from one to the other in the terminus region. The question today is: which is it? Replication that terminates at a specific site or replication that terminates by random collisions within the trap?
I am investigating this puzzle by developing a system wherein I can observe the location of termination of DNA replication directly in individual cells. I use Escherichia coli, an excellent model for bacterial study and a combination of bioinformatics, Green Fluorescent Protein (GFP) and fluorescent microscopy. This unique approach to understanding why the E. coli chromosome has the mutational patterns that it does can aid us in understanding DNA replication termination, a fundamental element in the cell cycle of this medically important bacterium. In addition, this set of novel and state of the art approaches are establishing the connection between the cell biology of replication and its effect on mutational patterns in chromosome sequences. Once verified, sequence based methods can be applied to all sequenced organisms in public databases that are difficult to directly experiment on. The patterns of mutation in the DNA can inform us about how cells naturally replicate.
We are also in the process of incorporating fluorescent tools to study the replication dynamics in the Pseudomonas fluorescens SBW25 strain.
Animation of the circular bacterial chromosome replicating. The replisome is depicted as a red complex and there are two acting at each replication fork as it proceeds from the origin towards to the terminus. The issues that we are currently investigating involve the location of the meeting of the replication forks.