Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication

Abstract

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I’s mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.

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

“…Replication of a neutral sequence by LF-Pol I generates random mutations whose distribution identifies Pol I templates with high resolution (Allen et al 2011). The main limitation of this polymerase template-mapping approach is our inability to identify the strand where mutations originally occurred.…”

“…In a previous article, we addressed this problem using biases in the distribution of complementary mutations to define probabilistic markers, i.e. markers that indicated a higher probability of originating in one strand vs. the other (Allen et al 2011). For each complementary pair, the most frequent mutation (A→G; C→T; A→T; G→T) was designated as a marker for leading-strand synthesis and the least frequent, complementary mutations (T→C; G→A; T→A; C→A), as markers for lagging-strand synthesis.…”

“…The present analysis includes two new libraries: one targeting GFP and another one targeting human ALKBH1. These libraries are of high quality because they underwent multiple rounds of mutagenesis (n=4), which increases the mutation density and facilitates the identification of Okazaki processing sites (Allen et al 2011), and because they involve sequences that are neutral, i.e. sequences that provide no significant fitness advantage or disadvantage to the host.…”

“…In a previous article (Allen et al 2011), a thorough analysis of the footprint of error-prone Pol I replication of ColE1 plasmids allowed us to establish the following: 1) that Pol I replication extends well beyond the ~100 nt of RNA II primer extension reported in vitro ; 2) we found the likely location of Okazaki processing sites on the lagging strand of the plasmid; 3) we estimated the extent of 5′→3′ exonuclease processing by Pol I at Okazaki processing sites to be ~20 nucleotides.…”

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

“…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). It is worth noting that LF-Pol I is partially dominant in vivo , as expression of this polymerase still produces Col E1 plasmid mutagenesis at permissive temperature or in polA WT strains, albeit with a 3- to 5-fold lower frequency relative to JS200 at restrictive temperature (28).…”