Single-molecule analysis of double-stranded DNA break repair in living bacteria

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Our study uses ultra-sensitive microscopes to observe important processes taking place during the repair of DNA, the molecule wherein the genetic information is stored mainly in the form of chromosomes. DNA repair is the general term for the collection of the different ways living cells fix different types of damage to their genetic material. As such, DNA repair is central for the survival and growth of all living organisms. Specifically, our work focuses on the process of repairing broken chromosomes when they are exposed to breaks on both strands of the DNA double helix; such breaks are known as "double-stranded breaks". In bacteria, these DNA breaks are often fixed by two protein machines called RecBCD and RecA, that act in a well coordinated fashion. The RecBCD machine manages to find the broken DNA ends amongst huge amounts of intact DNA in the cell and removes part of the broken DNA end to leave a special DNA structure. This structure is then recognised by many copies of the RecA machine that forms of stiff DNA filament that embarks on a fascinating and mysterious search for an intact copy of the chromosome. This process needs to be fast and specific, since any errors not fixed in time can lead to dangerous mutations or even cell death.Much of what we know about how DNA breaks are fixed comes from studies with purified proteins and DNA in the test tube; these involve simple mixtures of the RecBCD and RecA machines with DNA sequences and helper proteins that accelerate or facilitate the process. However, the mechanisms of DNA repair in actual living organisms can be very different, due to the myriad of other biological components that are present in cells, and due to the way that the genes are packaged in the "bacterial nucleoid", which is a tightly packed structure made of the bacterial DNA and some of its proteins. To provide an example of the complexity that characterises DNA repair in living cells, one can consider the task of RecA: to search just 50 letters of DNA within the entire chromosome, which is a molecule longer than 5,000,000 letters and highly folded. Another example of the complexity has to do with other proteins that can interfere with or facilitate the function of RecBCD when it tries to fix DNA ends that have been formed by treating pathogenic bacteria with antibiotics.To study the process of DNA repair in its natural environment of living cells, we will use advanced fluorescence microscopy to look at how the RecBCD machine searches, finds, and helps broken DNA ends with the help of its partner RecA inside living bacterial cells. We will use the bacterium Escherichia coli, which is a simple model organism for understanding biological mechanisms. A special feature of our work is that it is performed using a special microscope (a "single-molecule fluorescence microscope"). This microscope is carefully designed to allow detection and monitoring of individual (single) fluorescent molecules inside living cells (as opposed to conventional microscopes that require thousands or millions of fluorescent molecules).Using our powerful microscope to record movies and track the position of the proteins as they move, we will see how proteins search and find different types of broken DNA ends, either on the chromosome or on synthetic types of broken DNA that allow us to see the process more directly and in real time. We will also study how individual RecBCD machines cut out the DNA end to prepare the ground for the landing of the RecA machines. Finally, we will study how RecA forms the type of DNA that is able to search for the correct copy of the DNA to fix the damaged part. Our studies will improve our understanding of how DNA repair works in living cells, and help other scientists to study DNA repair in other organisms (such as humans), as well as to develop new pharmaceuticals that will improve the health of humans, animals and plants by disabling the DNA repair machinery of dangerous microbes.