E-Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

Live-cell single-molecule imaging of DNA Polymerase II: is the beta clamp required for recruitment? (#202)

Thomas Armstrong 1 2 , Megan E Cherry 1 2 , Elizabeth A Wood 3 , Phuong Pham 4 , Myron F Goodman 4 , Michael M Cox 3 , Antoine van Oijen 1 2 , Sarah S Henrikus 1 2 , Andrew Robinson 1 2
  1. Molecular Horizons Institute and School of Chemistry and Biomolecular Science, University of Wollongong, Wollongong, NSW, Australia
  2. Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia
  3. Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin, United States of America
  4. Departments of Biological Sciences and Chemistry, University of Southern California, Los Angles, California, United States of America

DNA polymerase II (Pol II) is one of three specialised polymerases to be upregulated following DNA damage in E. coli. Pol II differs from the other damage inducible polymerases (Pols IV and V), in that it belongs to the B family polymerases and contains a 3′-5′ proof-reading exonuclease activity. Many decades of study have to reveal a clear biological role for Pol II. To further investigate its potential biological roles we are using live cell single-molecule imaging.

We have observed that in the absence of exogenous DNA damage Pol II forms short-lived, transient foci, indicative of some housekeeping function. Pol II foci were seen to colocalise tightly and to a high extent (15-20% colocalise with replisomes). The canonical model of TLS would have us believe that Pol II acts at replisomes, particularly following DNA damage. However, we have seen that following an array of exogenous DNA damage (UV exposure, ciprofloxacin, and azidothymidine) that Pol II is rapidly removed from the replisome (colocalisation drops to ~5%). This finding not only goes against the traditional TLS model but also brings into question the role of the E. coli beta clamp.

It is known that the presence of beta greatly stimulates the activity of all Pol II, however, it remains to be seen whether the beta clamp is essential for all Pol II recruitment in live E. coli cells. To answer these questions we have created a fluorescent beta clamp to couple with our already studied fluorescent Pol II. This has enabled us to monitor the positions of Pol II and the beta clamp in the absence and presence of exogenous DNA damage. We are now assessing the extent to which Pol II colocalises with the beta clamp in the presence and absence of DNA damage.