DNA polymerase II (Pol II) is one of three specialised polymerases to be upregulated following DNA damage in E. coli. Pol II differs from other damage inducible polymerases (Pols IV and V), in that it belongs to the B family and includes 3′-5′ proof-reading exonuclease activity. Many decades of study have yet to reveal a clear biological role for Pol II. To gain further information into potential biological roles of Poll II, we have turned to live cell single-molecule imaging.
Previously, we have imaged Pols IV and V to reveal novel and unanticipated aspects of their activities. By imaging Pol II in cells, we have now completed the set. We observe that Pol II forms transient foci in the absence of exogenous DNA damage, indicative of short-lived DNA binding activity (~50 ms). This suggests that Pol II has a housekeeping function. Pol II foci were also seen to colocalise much more tightly with replication fork markers and to a higher extent than both Pols IV and V (15-20% colocalise with replisomes).
Significant changes in Pol II behaviour are observed in cells treated with the DNA damaging antibiotic ciprofloxacin. Within minutes of ciprofloxacin treatment, the colocalisation of Pol II with replisomes drops to baseline levels. Over the next 60 minutes colocalisation levels partly recover (~10%). Pol II foci behaviour over this time also changes, with foci becoming much longer lived (~1 s). We are currently investigating whether this change in behaviour stems from the polymerase or exonuclease. The damage-induced drop in colocalisation is lost in RecFOR mutants suggesting that Pol II acts on single-stranded DNA gaps behind the replication fork. Finally, experimental evolution measurements show cells lacking Pol II develop ciprofloxacin resistance faster than wild-type cells indicating a major activity of Pol II is proof reading in ciprofloxacin treated cells.