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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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Related in: MedlinePlus

OJ and polymorphism rates are elevated at yeast DNase I footprintsa, b, DNase I footprint edges correspond, genome-wide, to elevated OJ rates and locally elevated polymorphism rates in S. cerevisiae (a), a pattern that is maintained when footprints associated with Reb1 and Rap1 binding sites are excluded (b). Genome-wide DNase I footprints (n=6,063) and excluding those within 50 nt of a Reb1 or Rap1 binding site (n=5,136) were aligned to their midpoint. c, d, Aligning DNase I footprints on their left edge rather than midpoint (to compensate for substantial heterogeneity in footprint size) demonstrates a distinct shoulder of elevated polymorphism rate at the aligned edge (c), with a significant elevation compared to nearby sequence upstream from the footprint (d). DNase I footprints from a were aligned to their left edge (x=0) with corresponding polymorphism rates shown (c). The elevated polymorphism rate cannot be explained by local sequence compositional distortions (d). Nucleotide substitution rates in the 11 nt centred on the DNase footprint edge (pink line), and another 11 nt encompassing positions −35 to −25 relative to the footprint edge (green line) were quantified. Darker pink and green filled circles denote the mean of observed substitution rates and lighter shades denote the mean for the same sites after 3-nucleotide preserving genomic shuffles. Error bars, SD; Mann-Whitney test. e, Model: Correlation of increased nucleotide substitution and OJ rates are consistent with elevated mutation frequency across heterogeneous DNase I footprints. Polymorphism is reduced at sequence-specific binding sites within the footprints, due to functional constraint. Therefore the effect of OF-related mutagenesis in these regions is most sensitively detected in the region immediately adjacent to the binding site (left of vertical dashed blue line, representing footprints aligned to their left edge). This ‘shoulder’ of elevated nucleotide substitutions represents sites with elevated, OJ-associated mutation is followed by a region of depressed substitution rates, owing to selective effects of the functional binding sites within the footprints (to the right of the dashed blue line). Signals further to the right are not interpretable given the heterogeneity in DNase I footprint sizes. Given strong selection at TF and DNase I footprint sites, this ‘shoulder’ of elevated nucleotide substitutions could represent a measure for the local mutation rate for such regions, analogous to that measured by the 4-fold degenerate sites in protein coding sequence.
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Figure 12: OJ and polymorphism rates are elevated at yeast DNase I footprintsa, b, DNase I footprint edges correspond, genome-wide, to elevated OJ rates and locally elevated polymorphism rates in S. cerevisiae (a), a pattern that is maintained when footprints associated with Reb1 and Rap1 binding sites are excluded (b). Genome-wide DNase I footprints (n=6,063) and excluding those within 50 nt of a Reb1 or Rap1 binding site (n=5,136) were aligned to their midpoint. c, d, Aligning DNase I footprints on their left edge rather than midpoint (to compensate for substantial heterogeneity in footprint size) demonstrates a distinct shoulder of elevated polymorphism rate at the aligned edge (c), with a significant elevation compared to nearby sequence upstream from the footprint (d). DNase I footprints from a were aligned to their left edge (x=0) with corresponding polymorphism rates shown (c). The elevated polymorphism rate cannot be explained by local sequence compositional distortions (d). Nucleotide substitution rates in the 11 nt centred on the DNase footprint edge (pink line), and another 11 nt encompassing positions −35 to −25 relative to the footprint edge (green line) were quantified. Darker pink and green filled circles denote the mean of observed substitution rates and lighter shades denote the mean for the same sites after 3-nucleotide preserving genomic shuffles. Error bars, SD; Mann-Whitney test. e, Model: Correlation of increased nucleotide substitution and OJ rates are consistent with elevated mutation frequency across heterogeneous DNase I footprints. Polymorphism is reduced at sequence-specific binding sites within the footprints, due to functional constraint. Therefore the effect of OF-related mutagenesis in these regions is most sensitively detected in the region immediately adjacent to the binding site (left of vertical dashed blue line, representing footprints aligned to their left edge). This ‘shoulder’ of elevated nucleotide substitutions represents sites with elevated, OJ-associated mutation is followed by a region of depressed substitution rates, owing to selective effects of the functional binding sites within the footprints (to the right of the dashed blue line). Signals further to the right are not interpretable given the heterogeneity in DNase I footprint sizes. Given strong selection at TF and DNase I footprint sites, this ‘shoulder’ of elevated nucleotide substitutions could represent a measure for the local mutation rate for such regions, analogous to that measured by the 4-fold degenerate sites in protein coding sequence.

Mentions: Finally, to extend our analysis beyond common TF binding sites, we investigated whether the same mutational signature could be found for a broad range of regions at which regulatory proteins bind, regions we identified by the presence of DNase I footprints. Our preceding analysis of TFs suggested that nucleotide substitutions would be elevated immediately adjacent to the protein binding region defined by such footprints. In yeast we found that DNase I footprint edges served as a good proxy for elevated OJ rate with significantly elevated substitution rates (Extended data Fig. 7). Similarly, in humans, aligning regions containing DNase I footprints on the basis of boundary junctions (left-hand edge of footprint), detected substantially elevated nucleotide substitution rates close to the junction, relative to the baseline rate in the immediate region (Fig. 5d). These increased substitution rates were related to position rather than sequence content, as this signal was lost when a 3-mer preserving genome shuffle was applied, both for individual TFs (Fig. 5b; Extended data Fig. 6a-d) and DNase I footprints (Fig. 5d). Therefore this mutational signature is not due to the retention of mutagenic sequences (e.g. CpG dinucleotides) at such sites42, and is a widespread phenomenon in the genome at protein binding sites in both yeast and humans.


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)

OJ and polymorphism rates are elevated at yeast DNase I footprintsa, b, DNase I footprint edges correspond, genome-wide, to elevated OJ rates and locally elevated polymorphism rates in S. cerevisiae (a), a pattern that is maintained when footprints associated with Reb1 and Rap1 binding sites are excluded (b). Genome-wide DNase I footprints (n=6,063) and excluding those within 50 nt of a Reb1 or Rap1 binding site (n=5,136) were aligned to their midpoint. c, d, Aligning DNase I footprints on their left edge rather than midpoint (to compensate for substantial heterogeneity in footprint size) demonstrates a distinct shoulder of elevated polymorphism rate at the aligned edge (c), with a significant elevation compared to nearby sequence upstream from the footprint (d). DNase I footprints from a were aligned to their left edge (x=0) with corresponding polymorphism rates shown (c). The elevated polymorphism rate cannot be explained by local sequence compositional distortions (d). Nucleotide substitution rates in the 11 nt centred on the DNase footprint edge (pink line), and another 11 nt encompassing positions −35 to −25 relative to the footprint edge (green line) were quantified. Darker pink and green filled circles denote the mean of observed substitution rates and lighter shades denote the mean for the same sites after 3-nucleotide preserving genomic shuffles. Error bars, SD; Mann-Whitney test. e, Model: Correlation of increased nucleotide substitution and OJ rates are consistent with elevated mutation frequency across heterogeneous DNase I footprints. Polymorphism is reduced at sequence-specific binding sites within the footprints, due to functional constraint. Therefore the effect of OF-related mutagenesis in these regions is most sensitively detected in the region immediately adjacent to the binding site (left of vertical dashed blue line, representing footprints aligned to their left edge). This ‘shoulder’ of elevated nucleotide substitutions represents sites with elevated, OJ-associated mutation is followed by a region of depressed substitution rates, owing to selective effects of the functional binding sites within the footprints (to the right of the dashed blue line). Signals further to the right are not interpretable given the heterogeneity in DNase I footprint sizes. Given strong selection at TF and DNase I footprint sites, this ‘shoulder’ of elevated nucleotide substitutions could represent a measure for the local mutation rate for such regions, analogous to that measured by the 4-fold degenerate sites in protein coding sequence.
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Related In: Results  -  Collection

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Figure 12: OJ and polymorphism rates are elevated at yeast DNase I footprintsa, b, DNase I footprint edges correspond, genome-wide, to elevated OJ rates and locally elevated polymorphism rates in S. cerevisiae (a), a pattern that is maintained when footprints associated with Reb1 and Rap1 binding sites are excluded (b). Genome-wide DNase I footprints (n=6,063) and excluding those within 50 nt of a Reb1 or Rap1 binding site (n=5,136) were aligned to their midpoint. c, d, Aligning DNase I footprints on their left edge rather than midpoint (to compensate for substantial heterogeneity in footprint size) demonstrates a distinct shoulder of elevated polymorphism rate at the aligned edge (c), with a significant elevation compared to nearby sequence upstream from the footprint (d). DNase I footprints from a were aligned to their left edge (x=0) with corresponding polymorphism rates shown (c). The elevated polymorphism rate cannot be explained by local sequence compositional distortions (d). Nucleotide substitution rates in the 11 nt centred on the DNase footprint edge (pink line), and another 11 nt encompassing positions −35 to −25 relative to the footprint edge (green line) were quantified. Darker pink and green filled circles denote the mean of observed substitution rates and lighter shades denote the mean for the same sites after 3-nucleotide preserving genomic shuffles. Error bars, SD; Mann-Whitney test. e, Model: Correlation of increased nucleotide substitution and OJ rates are consistent with elevated mutation frequency across heterogeneous DNase I footprints. Polymorphism is reduced at sequence-specific binding sites within the footprints, due to functional constraint. Therefore the effect of OF-related mutagenesis in these regions is most sensitively detected in the region immediately adjacent to the binding site (left of vertical dashed blue line, representing footprints aligned to their left edge). This ‘shoulder’ of elevated nucleotide substitutions represents sites with elevated, OJ-associated mutation is followed by a region of depressed substitution rates, owing to selective effects of the functional binding sites within the footprints (to the right of the dashed blue line). Signals further to the right are not interpretable given the heterogeneity in DNase I footprint sizes. Given strong selection at TF and DNase I footprint sites, this ‘shoulder’ of elevated nucleotide substitutions could represent a measure for the local mutation rate for such regions, analogous to that measured by the 4-fold degenerate sites in protein coding sequence.
Mentions: Finally, to extend our analysis beyond common TF binding sites, we investigated whether the same mutational signature could be found for a broad range of regions at which regulatory proteins bind, regions we identified by the presence of DNase I footprints. Our preceding analysis of TFs suggested that nucleotide substitutions would be elevated immediately adjacent to the protein binding region defined by such footprints. In yeast we found that DNase I footprint edges served as a good proxy for elevated OJ rate with significantly elevated substitution rates (Extended data Fig. 7). Similarly, in humans, aligning regions containing DNase I footprints on the basis of boundary junctions (left-hand edge of footprint), detected substantially elevated nucleotide substitution rates close to the junction, relative to the baseline rate in the immediate region (Fig. 5d). These increased substitution rates were related to position rather than sequence content, as this signal was lost when a 3-mer preserving genome shuffle was applied, both for individual TFs (Fig. 5b; Extended data Fig. 6a-d) and DNase I footprints (Fig. 5d). Therefore this mutational signature is not due to the retention of mutagenic sequences (e.g. CpG dinucleotides) at such sites42, and is a widespread phenomenon in the genome at protein binding sites in both yeast and humans.

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