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Lagging-strand replication shapes the mutational landscape of the genome.

Reijns MA, Kemp H, Ding J, de Procé SM, Jackson AP, Taylor MS - Nature (2015)

Bottom Line: The origin of mutations is central to understanding evolution and of key relevance to health.Here we report that the 5' ends of Okazaki fragments have significantly increased levels of nucleotide substitution, indicating a replicative origin for such mutations.Using a novel method, emRiboSeq, we map the genome-wide contribution of polymerases, and show that despite Okazaki fragment processing, DNA synthesized by error-prone polymerase-α (Pol-α) is retained in vivo, comprising approximately 1.5% of the mature genome.

View Article: PubMed Central - PubMed

Affiliation: Medical and Developmental Genetics, MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.

ABSTRACT
The origin of mutations is central to understanding evolution and of key relevance to health. Variation occurs non-randomly across the genome, and mechanisms for this remain to be defined. Here we report that the 5' ends of Okazaki fragments have significantly increased levels of nucleotide substitution, indicating a replicative origin for such mutations. Using a novel method, emRiboSeq, we map the genome-wide contribution of polymerases, and show that despite Okazaki fragment processing, DNA synthesized by error-prone polymerase-α (Pol-α) is retained in vivo, comprising approximately 1.5% of the mature genome. We propose that DNA-binding proteins that rapidly re-associate post-replication act as partial barriers to Pol-δ-mediated displacement of Pol-α-synthesized DNA, resulting in incorporation of such Pol-α tracts and increased mutation rates at specific sites. We observe a mutational cost to chromatin and regulatory protein binding, resulting in mutation hotspots at regulatory elements, with signatures of this process detectable in both yeast and humans.

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Mapping replicative polymerase DNA synthesis using emRiboSeqa, Point mutations in replicative polymerases elevate ribonucleotide incorporation rates, permitting their contribution to genome synthesis to be tracked. Schematic of replication fork with polymerases and their ribonucleotide incorporation rates (27,30 and JS Williams, AR Clausen & TA Kunkel, personal communication) as indicated (POL, WT polymerases; *, point mutants). Embedded ribonucleotides indicated by ‘R’; additional incorporation events due to polymerase mutations highlighted by shaded circles. b, c, Mapping of leading/lagging strand synthesis by Pol δ* and Pol ε* yeast strain using emRiboSeq (as in Fig. 3) highlights both experimentally validated (pink dotted lines) and putative replication origins (grey dotted lines). These often correspond to regions of early replicating DNA36 (c). d, Pol α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand in stationary phase yeast, as shown by the opposite pattern for a polymerase WT strain. Strand ratios are shown as best fit splines with 80 degrees of freedom, y-axes show log2 of the strand ratio calculated in 2,001 nt windows (b-d).
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Figure 8: Mapping replicative polymerase DNA synthesis using emRiboSeqa, Point mutations in replicative polymerases elevate ribonucleotide incorporation rates, permitting their contribution to genome synthesis to be tracked. Schematic of replication fork with polymerases and their ribonucleotide incorporation rates (27,30 and JS Williams, AR Clausen & TA Kunkel, personal communication) as indicated (POL, WT polymerases; *, point mutants). Embedded ribonucleotides indicated by ‘R’; additional incorporation events due to polymerase mutations highlighted by shaded circles. b, c, Mapping of leading/lagging strand synthesis by Pol δ* and Pol ε* yeast strain using emRiboSeq (as in Fig. 3) highlights both experimentally validated (pink dotted lines) and putative replication origins (grey dotted lines). These often correspond to regions of early replicating DNA36 (c). d, Pol α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand in stationary phase yeast, as shown by the opposite pattern for a polymerase WT strain. Strand ratios are shown as best fit splines with 80 degrees of freedom, y-axes show log2 of the strand ratio calculated in 2,001 nt windows (b-d).

Mentions: Control experiments using endonucleases of known sequence specificity demonstrated 99.9% strand specificity and 99.9% site specificity for the technique (Extended data Fig. 2b-d). Using RNase H2 deficient Pol-ε M644G and Pol-δ L612M yeast strains we then mapped the relative contributions of these respective polymerases genome-wide (Fig. 3c-e; Extended data Fig. 3). We found that ribonucleotide incorporation in the Pol-δ L612M strain was substantially enriched on the DNA strand that is preferentially synthesised by lagging strand synthesis17, in keeping with its function as the major lagging strand polymerase30,33,34, while ribonucleotide incorporation in the Pol-ε M644G strain exhibited an entirely reciprocal pattern consistent with its function as the leading strand polymerase31,35 (Fig. 3e). Furthermore, points at which neither enzyme showed strand preference (intersection of both Pol-ε and Pol-δ plots with the x-axis) corresponded precisely with annotated origins of replication. Other intersection points were also evident that correspond to replication termination regions, as well as putative, non-annotated origins. The latter overlapped with early replicating regions36 (Extended data Fig. 3b-c). Therefore we concluded that emRiboSeq can be used to determine the distribution of polymerase activity genome-wide, and has utility for the identification of replication origin and termination sites.


Lagging-strand replication shapes the mutational landscape of the genome.

Reijns MA, Kemp H, Ding J, de Procé SM, Jackson AP, Taylor MS - Nature (2015)

Mapping replicative polymerase DNA synthesis using emRiboSeqa, Point mutations in replicative polymerases elevate ribonucleotide incorporation rates, permitting their contribution to genome synthesis to be tracked. Schematic of replication fork with polymerases and their ribonucleotide incorporation rates (27,30 and JS Williams, AR Clausen & TA Kunkel, personal communication) as indicated (POL, WT polymerases; *, point mutants). Embedded ribonucleotides indicated by ‘R’; additional incorporation events due to polymerase mutations highlighted by shaded circles. b, c, Mapping of leading/lagging strand synthesis by Pol δ* and Pol ε* yeast strain using emRiboSeq (as in Fig. 3) highlights both experimentally validated (pink dotted lines) and putative replication origins (grey dotted lines). These often correspond to regions of early replicating DNA36 (c). d, Pol α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand in stationary phase yeast, as shown by the opposite pattern for a polymerase WT strain. Strand ratios are shown as best fit splines with 80 degrees of freedom, y-axes show log2 of the strand ratio calculated in 2,001 nt windows (b-d).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374164&req=5

Figure 8: Mapping replicative polymerase DNA synthesis using emRiboSeqa, Point mutations in replicative polymerases elevate ribonucleotide incorporation rates, permitting their contribution to genome synthesis to be tracked. Schematic of replication fork with polymerases and their ribonucleotide incorporation rates (27,30 and JS Williams, AR Clausen & TA Kunkel, personal communication) as indicated (POL, WT polymerases; *, point mutants). Embedded ribonucleotides indicated by ‘R’; additional incorporation events due to polymerase mutations highlighted by shaded circles. b, c, Mapping of leading/lagging strand synthesis by Pol δ* and Pol ε* yeast strain using emRiboSeq (as in Fig. 3) highlights both experimentally validated (pink dotted lines) and putative replication origins (grey dotted lines). These often correspond to regions of early replicating DNA36 (c). d, Pol α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand in stationary phase yeast, as shown by the opposite pattern for a polymerase WT strain. Strand ratios are shown as best fit splines with 80 degrees of freedom, y-axes show log2 of the strand ratio calculated in 2,001 nt windows (b-d).
Mentions: Control experiments using endonucleases of known sequence specificity demonstrated 99.9% strand specificity and 99.9% site specificity for the technique (Extended data Fig. 2b-d). Using RNase H2 deficient Pol-ε M644G and Pol-δ L612M yeast strains we then mapped the relative contributions of these respective polymerases genome-wide (Fig. 3c-e; Extended data Fig. 3). We found that ribonucleotide incorporation in the Pol-δ L612M strain was substantially enriched on the DNA strand that is preferentially synthesised by lagging strand synthesis17, in keeping with its function as the major lagging strand polymerase30,33,34, while ribonucleotide incorporation in the Pol-ε M644G strain exhibited an entirely reciprocal pattern consistent with its function as the leading strand polymerase31,35 (Fig. 3e). Furthermore, points at which neither enzyme showed strand preference (intersection of both Pol-ε and Pol-δ plots with the x-axis) corresponded precisely with annotated origins of replication. Other intersection points were also evident that correspond to replication termination regions, as well as putative, non-annotated origins. The latter overlapped with early replicating regions36 (Extended data Fig. 3b-c). Therefore we concluded that emRiboSeq can be used to determine the distribution of polymerase activity genome-wide, and has utility for the identification of replication origin and termination sites.

Bottom Line: The origin of mutations is central to understanding evolution and of key relevance to health.Here we report that the 5' ends of Okazaki fragments have significantly increased levels of nucleotide substitution, indicating a replicative origin for such mutations.Using a novel method, emRiboSeq, we map the genome-wide contribution of polymerases, and show that despite Okazaki fragment processing, DNA synthesized by error-prone polymerase-α (Pol-α) is retained in vivo, comprising approximately 1.5% of the mature genome.

View Article: PubMed Central - PubMed

Affiliation: Medical and Developmental Genetics, MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XU, UK.

ABSTRACT
The origin of mutations is central to understanding evolution and of key relevance to health. Variation occurs non-randomly across the genome, and mechanisms for this remain to be defined. Here we report that the 5' ends of Okazaki fragments have significantly increased levels of nucleotide substitution, indicating a replicative origin for such mutations. Using a novel method, emRiboSeq, we map the genome-wide contribution of polymerases, and show that despite Okazaki fragment processing, DNA synthesized by error-prone polymerase-α (Pol-α) is retained in vivo, comprising approximately 1.5% of the mature genome. We propose that DNA-binding proteins that rapidly re-associate post-replication act as partial barriers to Pol-δ-mediated displacement of Pol-α-synthesized DNA, resulting in incorporation of such Pol-α tracts and increased mutation rates at specific sites. We observe a mutational cost to chromatin and regulatory protein binding, resulting in mutation hotspots at regulatory elements, with signatures of this process detectable in both yeast and humans.

Show MeSH
Related in: MedlinePlus