UC Santa Cruz

ColE1 plasmid replication is unidirectional and requires two DNA polymerases: DNA polymerase I (Pol I) and DNA polymerase III (Pol III). Pol I initiates leading-strand synthesis by extending an RNA primer, allowing the Pol III holoenzyme to assemble and finish replication of both strands. The goal of the present work is to study the interplay between Pol I and Pol III during ColE1 plasmid replication, to gain new insights into Pol I function in vivo. Our approach consists of using mutations generated by a low-fidelity mutant of Pol I (LF-Pol I) during replication of a ColE1 plasmid as a footprint for Pol I replication. This approach allowed mapping areas of Pol I replication on the plasmid with high resolution. In addition, we were able to approximate the strandedness of Pol I mutations throughout the plasmid, allowing us to estimate the spectrum of the LF-Pol I in vivo. Our study produced the following three mechanistic insights: (1) we identified the likely location of the polymerase switch at ~200 bp downstream of replication initiation; (2) we found evidence suggesting that Pol I can replicate both strands, supporting earlier studies indicating a functional redundancy between Pol I and Pol III (3) we found evidence pointing to a specific role of Pol I during termination of lagging-strand replication. In addition, we illustrate how our strand-specific footprinting approach can be used to dissect factors modulating Pol I fidelity in vivo.