Single-molecule studies of exonuclease proteins

DNA replication and maintenance are an integral part of the continued survival of all life on this planet. Therefore, the collection of proteins and the mechanisms they employ to maintain and copy DNA in the presence of continuous damage experienced from everyday life is vital. Although these proteins have been rigorously studied in their structure and function, there is still more to know and understand. Exonuclease proteins perform an essential role during DNA replication and repair, with analogues existing in every organism. They act as a source of proofreading during replication, play a role in repairing DNA damage and prepare the DNA for recombination. Exonucleases have been studied extensively over the last couple decades in using primarily bulk ensemble assays, from which a significant amount of important information has been gathered. However, bulk ensemble assays can only observe the average behaviour of large numbers of proteins and cannot observe the behaviour of single proteins directly. This study focused on investigating the activity of exonuclease proteins on DNA at a single-molecule level using primarily forced-based techniques. The aim of this project was to establish single-molecule assays to visualise the kinetics with which different exonucleases digest DNA. The processivity and digestion rate of three different exonucleases were studied during the studies described here (E. coli Exonuclease III, E. coli Polymerase III, and E. coli Exonuclease V) using a single-molecule hydrodynamic stretching assay that measures the length of DNA coupled to a bead. Results are consistent with previously reported bulk biochemistry studies showing the highly distributive nature of Exonuclease III, with Polymerase III and Exonuclease V being far more processive. This body of work forms a basis for future work into the mechanisms of action of exonuclease proteins, with an opportunity to focus on the effects of physical forces on the enzymatic activity.