Replication repair in real life: analysing how broken DNA replication machines are rebuilt inside cells.

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DNA encodes the genetic blueprint that provides the basis for all life. Any corruption of this blueprint can have very harmful consequences for the organism since it leads to mutations that can alter how cells function or even lead to death. In humans such mutations can lead to genetic diseases, cancer and possibly the debilitating effects of old age. Every time a cell divides it must copy its DNA so that each daughter cell receives an accurate, complete set of genetic instructions but unfortunately this copying process also has the potential to corrupt the blueprint. This corruption can occur because the nanomachines that duplicate DNA continually encounter unavoidable obstacles to their movement. Many of these obstacles can be cleared or bypassed, allowing the original nanomachine to continue on its way. However, some of these obstacles do lead to breakdown of the nanomachine. These breakdowns are potentially disastrous since they may lead to parts of the genetic code not being copied or miscopied prior to cell division, resulting in corruption of the code.These breakdowns, and the risks associated with them, are a problem that all organisms must face. Consequently mechanisms have evolved to reassemble the copying nanomachines back onto the DNA, allowing copying to resume and so reducing the risk of mistakes being introduced into the genetic code. The means by which these replicating nanomachines are reloaded back onto chromosomes appear to be similar in all organisms, a reflection of the conserved nature of the genetic material and the machines that copy this material. However, it is only in bacteria that the enzymes responsible for reloading these nanomachines back onto DNA have been identified. The bacterium Escherichia coli has provided a great deal of information about how this reloading occurs and has provided a model with which to understand how this repair occurs in all organisms. However, in spite of two decades of study, we still know very little about how this repair actually occurs inside cells. This ignorance is in part because of the many different types of obstacle encountered inside cells that may lead to breakdown of these nanomachines and also the complicated, overlapping nature of this nanomachine repair.To address this gap in our understanding of how the copying of DNA is achieved inside cells, we will use recently-developed microscopy techniques that allow single molecules to be visualised and tracked inside living cells. We will exploit the information available in the bacterium E. coli to monitor nanomachine repair as it happens inside cells. This information will allow us to generate a model of how the rebuilding of copying machines underpins duplication of the genetic blueprint in this simple system, providing insight into how this essential repair occurs inside more complicated organisms such as ourselves.