The mutagenic footprint of low-fidelity Pol I ColE1 plasmid replication in E. coli reveals an extensive interplay between Pol I and Pol III

Abstract

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 to 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, in order 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.

Fluorescence-Based Reporters for Detection of Mutagenesis in E. coli

“…(19). Compared with the profile that our group generated previously based on sequencing (32), only one pair exhibits a lower reversion frequency than expected: A:T→G:C. This could be the result of sequence-context dependent effects (37,38), which can be in part due to differential efficiency of mismatch repair (6). Our observation that S70R1, which detects A:T→C:G and A:T→T:A mutations, produces fewer reversions than S70T, which detects A:T→T:A alone directly confirms the impact of local sequence context on mutation rates.…”

“…Shift of this strain to 37 °C makes J200 cells dependent on the activity of LF-Pol I for survival. Pol I performs ColE1 plasmid replication (32) and processes of Okazaki fragments during lagging-strand replication in plasmid and chromosomal DNA (30,32). …”

“…This is true for most of the plasmid sequence, where Pol I appears to be competing with Pol III (32). These loads are higher in areas replicated exclusively by Pol I: the 150 nucleotides immediately downstream of the RNA/DNA switch (leading-strand synthesis by Pol I), ~500 nucleotides upstream of the RNA/DNA switch (gap-filling of lagging-strand synthesis by Pol I), and ~20nt patches corresponding to areas of Okazaki fragment processing by Pol I (29,33).…”

“…In terms of mutation spectrum, we were able to estimate the mutation frequency of LF-Pol I on a single strand in vivo (32). The vast majority of mutations (>95%) are point mutations and can be grouped in four groups: most frequent: C→T transitions (60%); frequent: A→G and A →T (20 and 10% of the total, respectively); rare: G→T, G →A, and G→T, and extremely rare: T→C, T→A, A→C and C→G.…”

“…The vast majority of mutations (>95%) are point mutations and can be grouped in four groups: most frequent: C→T transitions (60%); frequent: A→G and A →T (20 and 10% of the total, respectively); rare: G→T, G →A, and G→T, and extremely rare: T→C, T→A, A→C and C→G. Note that, based on the very low frequency of T→C transitions in vivo , mismatch repair appears to be intact in these cells (32). Given that our reporter detects mutations in double-stranded DNA, i.e .…”