Final Report Summary - POLMACHINE (Single-molecule analysis of DNA polymerase in vitro and in vivo: a machine in action) DNA polymerases are dynamic molecular machines that faithfully copy genetic information during DNA replication and DNA repair. DNA polymerases use the information in a DNA template to synthesize a DNA copy by adding nucleotides to the 3’-end of a DNA primer. Extensive crystallographic, biochemical and genetic analysis of DNA polymerase led to proposals of a general scheme for nucleotide addition. However, the approaches left many unanswered questions regarding polymerase mechanisms; these questions required the use of complementary single-molecule approaches, since such approaches remove complications due to ensemble- and time-averaging of observed signals, and provide unique capabilities for real-time monitoring of reaction, even inside living cells.During the POLMACHINE project, we developed novel fluorescence approaches to study DNA synthesis by a proofreading DNA polymerase (Pol I) through direct, real-time observation of its movements and interactions at the single-molecule level. We studied mechanisms of fidelity through comparisons involving mutant polymerases, mispaired nucleotides, or partially mispaired DNA; we will also studied the conformation and subcellular localisation of Pol I and its substrates inside bacterial cells. The project led to achievements in the understanding of the mechanisms of DNA polymerase I and nucleic-acids polymerases and DNA-processing machinery in general; it also led to the development of general methods and instruments that help the monitoring of biomolecular structure, conformational changes, and reaction kinetics both in vitro and inside living cells. Using a family of single-molecule methods based on fluorescence resonance energy transfer (FRET) and alternating-laser excitation (ALEX) microscopy, we showed how DNA polymerase I (Pol I) selects the appropriate substrates with extremely high fidelity and how different side chains control different stages in the fingers-closing transition that precedes the chemical step and controls fidelity. We also used these advanced FRET methods to measure >70 distance restraints within a Pol-I complex with gapped DNA (a crucial DNA-repair intermediate), and combined them with molecular modelling to construct a high-resolution view of the complex, providing unique insight on how the DNA gap is recognized and how it is deformed to facilitate nick translation by Pol I.We have also developed powerful ways to explore the mechanisms of DNA repair by studying the motion of proteins using single-molecule tracking inside living cells. By tracking the motions of single protein molecules as a function of time, we constructed trajectories that capture the diffusion profile of these molecules. We have used this method to study molecules of Pol I as they search and repair damaged DNA, and to measure the time for DNA-repair DNA synthesis in living E.coli cells. This is an important contribution, since in vitro studies, despite their exquisite level of control and high sensitivity, can only hint at actual reaction kinetics in living cells. Our measurements established a global view for key DNA-repair proteins that can be used to construct more reliable predictive theoretical models of DNA damage and repair in cells. Our study also offers a general tool that allows study of intracellular diffusion of any protein, regardless of how many copies it has in a cell. Our group also pioneered the use of electroporation to introduce fluorescent DNA, RNA, and proteins into bacteria and other microorganisms. This unique capability brings the stability and brightness of organic fluorophores inside living cells, thus enabling molecule tracking in the minute timescale. Further, the use of doubly-labelled electroporated molecules enabled the first single-molecule FRET study in the bacterial cytoplasm, opening the way for monitoring conformations, conformational changes and nanoscale distances inside living cells. Our work on developing instrumentation to pursue studies of DNA polymerase led us to the development of the Nanoimager, a versatile, robust, desktop single-molecule imaging and super-resolution microscope for the biological community and the biotechnology industry. The instrument promises to substantially increase the impact of single-molecule methods in academia and industry.
