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 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.
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Acknowledgments
The authors would like to thank Dr. Rachel Karchin for recruiting JY and YL to this project through her Computational Biology class, Cherie Musgrove for careful proofreading of an early version of the manuscript, and Drs. Thomas Kunkel and Roel Schaaper for critical input on the manuscript ahead of publication. This project was partially supported through a K08 award of the NIH (CA116429-04) to MC.
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Communicated by S. Hohmann.
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Suppl. Table 2 Sequence of the mutation hotspots. The sequence is listed in the 5′ to 3′ direction, with three flanking nucleotides on each side. Contiguous residues having at least one mutation are underlined, and positions having more than one mutation are highlighted in bold. The mutations found in each of the hotspots are listed to the right of the hotspot sequence. The “5′-CCA/TA/T-3′” motif is highlighted with a gray box. Hotspots for the GFP libraries (merged sequence of liquid and solid libraries) are shown on the left four columns. Mutations generated in liquid culture are shown in gray and mutations generated in colonies in black. Hotspots for the ALKBH1 libraries are shown on the right four columns (DOCX 154 kb)
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Suppl. Fig. 1. Identification of mutation hotspots through clustering analysis. Hotspot mutation index, defined as the number of consecutive mutations that can be found at a distance of ≤ 1 nucleotides from each other is shown on the y-axis relative to the distance (in nucleotides) of mutant positions from the RNA/DNA switch (x-axis). a GFP libraries. All 501 mutations from our 3 GFP libraries were included. The average distance between the 337 mutant positions was 2.52 nucleotides. b ALKBH1 libraries. All 257 mutations were included. The average distance between the 170 positions was 2.75 nucleotides (TIFF 1520 kb)
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Suppl. Fig. 2. Distribution of hotspot indices shown in Suppl. Fig. 1 for the GFP libraries (a) and for the ALKBH1 library (b). These indices (y-axis) quantify the number of mutations found in the same or adjacent position for given nucleotide positions (TIFF 1520 kb)
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Suppl. Fig. 3. Identification of putative Okazaki primer processing sites. a Clustering analysis. Number of consecutive positions with signature lagging-strand mutations that can be found at a distance of ≤ 5 nucleotides from each other (y-axis) relative to the distance (in nucleotides) of mutant positions from the RNA/DNA switch (x-axis). The average distance between lagging-strand mutant positions was 7.39 nucleotides. Clusters considered significant (n > 5 mutations) are labeled in roman numerals. The nucleotide positions for these sites are: 179-191; 559-578; 910-926; and 1035-1045 b Lagging-strand mutation enrichment. Mutations within the sites defined by clustering analysis (roman numerals on x-axis) were classified as signature leading or lagging strand as described in the methods and their ratio calculated. Columns represent ratio of lagging vs. leading at each of these sites (TIFF 1520 kb)
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Troll, C., Yoder, J., Alexander, D. et al. The mutagenic footprint of low-fidelity Pol I ColE1 plasmid replication in E. coli reveals an extensive interplay between Pol I and Pol III. Curr Genet 60, 123–134 (2014). https://doi.org/10.1007/s00294-013-0415-9
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DOI: https://doi.org/10.1007/s00294-013-0415-9