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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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Frequent nucleotide substitutions at OF 5′-endsa, Mutation rates are elevated downstream of OJs. Substitution polymorphisms (red) and OJ rate (blue) in regions surrounding high frequency OJs (top 0.1%). n=5,660 sequences orientated for dominant direction of OF synthesis. b, Mutation rates correlate with OJ peak size. Mutations are significantly enriched downstream of the junction (pink), compared to genome shuffle controls (light green/pink). Sites grouped by OJ frequency. Error bars, SD; paired two-sided t-test. c, Hypothesis: DNA synthesised by non-proofreading Pol-α is preferentially trapped in regions rapidly bound by proteins post-replication. These act as partial barriers to Pol-δ displacement of Pol-α synthesised DNA, resulting in locally elevated mutations.
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Figure 2: Frequent nucleotide substitutions at OF 5′-endsa, Mutation rates are elevated downstream of OJs. Substitution polymorphisms (red) and OJ rate (blue) in regions surrounding high frequency OJs (top 0.1%). n=5,660 sequences orientated for dominant direction of OF synthesis. b, Mutation rates correlate with OJ peak size. Mutations are significantly enriched downstream of the junction (pink), compared to genome shuffle controls (light green/pink). Sites grouped by OJ frequency. Error bars, SD; paired two-sided t-test. c, Hypothesis: DNA synthesised by non-proofreading Pol-α is preferentially trapped in regions rapidly bound by proteins post-replication. These act as partial barriers to Pol-δ displacement of Pol-α synthesised DNA, resulting in locally elevated mutations.

Mentions: The synthesis and processing of OFs is directional. Therefore substitution rates would be expected to be asymmetrical relative to the direction of synthesis, if a component of this process was the cause. As most of the genome is preferentially replicated with either the forward or reverse strand as the lagging strand, we orientated regions by their dominant direction of lagging strand synthesis. This revealed substantially elevated nucleotide substitution rates immediately downstream of OJs (Fig. 2a), the level of mutational signal correlating with OJ site frequency. Quantification of substitution rates for the five nucleotides immediately upstream and downstream of the OJ (Fig. 2b), demonstrated that high frequency OJ sites (11-fold elevated OJ rate relative to baseline; top 99.9 centile of sites) displayed the highest substitution rate (p < 2.2·10−16), with significant elevation (p < 2.2·10−16) for medium frequency sites, (6.1-fold, 99-99.9 centile) but not low frequency sites (p = 0.3, 1.7-fold, OJ sites <99 centile). This was not due to site-specific sequence biases, as the increase in substitution rate was lost after a 3-mer preserving genome shuffle. Therefore point mutations are enriched at the 5′ ends of mature OFs of frequently occurring OJ sites, sites that correspond to protein barriers to Pol-δ processivity17.


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)

Frequent nucleotide substitutions at OF 5′-endsa, Mutation rates are elevated downstream of OJs. Substitution polymorphisms (red) and OJ rate (blue) in regions surrounding high frequency OJs (top 0.1%). n=5,660 sequences orientated for dominant direction of OF synthesis. b, Mutation rates correlate with OJ peak size. Mutations are significantly enriched downstream of the junction (pink), compared to genome shuffle controls (light green/pink). Sites grouped by OJ frequency. Error bars, SD; paired two-sided t-test. c, Hypothesis: DNA synthesised by non-proofreading Pol-α is preferentially trapped in regions rapidly bound by proteins post-replication. These act as partial barriers to Pol-δ displacement of Pol-α synthesised DNA, resulting in locally elevated mutations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4374164&req=5

Figure 2: Frequent nucleotide substitutions at OF 5′-endsa, Mutation rates are elevated downstream of OJs. Substitution polymorphisms (red) and OJ rate (blue) in regions surrounding high frequency OJs (top 0.1%). n=5,660 sequences orientated for dominant direction of OF synthesis. b, Mutation rates correlate with OJ peak size. Mutations are significantly enriched downstream of the junction (pink), compared to genome shuffle controls (light green/pink). Sites grouped by OJ frequency. Error bars, SD; paired two-sided t-test. c, Hypothesis: DNA synthesised by non-proofreading Pol-α is preferentially trapped in regions rapidly bound by proteins post-replication. These act as partial barriers to Pol-δ displacement of Pol-α synthesised DNA, resulting in locally elevated mutations.
Mentions: The synthesis and processing of OFs is directional. Therefore substitution rates would be expected to be asymmetrical relative to the direction of synthesis, if a component of this process was the cause. As most of the genome is preferentially replicated with either the forward or reverse strand as the lagging strand, we orientated regions by their dominant direction of lagging strand synthesis. This revealed substantially elevated nucleotide substitution rates immediately downstream of OJs (Fig. 2a), the level of mutational signal correlating with OJ site frequency. Quantification of substitution rates for the five nucleotides immediately upstream and downstream of the OJ (Fig. 2b), demonstrated that high frequency OJ sites (11-fold elevated OJ rate relative to baseline; top 99.9 centile of sites) displayed the highest substitution rate (p < 2.2·10−16), with significant elevation (p < 2.2·10−16) for medium frequency sites, (6.1-fold, 99-99.9 centile) but not low frequency sites (p = 0.3, 1.7-fold, OJ sites <99 centile). This was not due to site-specific sequence biases, as the increase in substitution rate was lost after a 3-mer preserving genome shuffle. Therefore point mutations are enriched at the 5′ ends of mature OFs of frequently occurring OJ sites, sites that correspond to protein barriers to Pol-δ processivity17.

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