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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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Quantification of in vivo ribonucleotide incorporation by replicative polymerasesa, b, Representative alkaline gel electrophoresis of genomic DNA from yeast strains with mutant replicative DNA polymerases (a), with accompanying densitometry plots (b). Embedded ribonucleotides are detected by increased fragmentation of genomic DNA following alkaline treatment in an RNase H2-deficient (Δrnh201) background. Elevated rates are seen with all three mutant polymerases (indicated by *, as defined in Extended data Fig. 3a), and are reduced in Pol-ε′ which contains the point mutation M664L, a mutation that increases selectivity for dNTPs over rNTPs27. c, Quantification of average ribonucleotide incorporation in polymerase mutants from n=4 independent experiments. DNA isolated from mid-log phase cultures; error bars, SE. Overall ribonucleotide content is the product of incorporation frequency and the total contribution of each polymerase, resulting in the total ribonucleotide content detected to be highest for Pol-ε* (14,200 per genome), followed by Pol-δ* (4,300 per genome), Pol-α* (2,700 per genome), POL (1,900 per genome) and Pol-ε′ (860 per genome). d, The majority of the yeast genome exhibits directional asymmetry in replication (median 4:1 strand ratio). Count of genomic segments calculated for consecutive 2,001 nt windows over the yeast genome based on reanalysis of OF sequencing data17 denoted as ‘Okazaki-seq’. The strand asymmetry ratio was calculated after re-orienting all regions such that the predominant lagging strand was the forward strand. e-g, Genome-wide quantification of strand-specific incorporation of wild type and mutant replicative DNA polymerases determined by emRiboSeq reflects their roles in leading and lagging strand replication. A close to linear correlation with Okazaki-seq strand ratios is observed. The strand ratio preference for lagging strand ribonucleotide incorporation for independent libraries (including stationary phase libraries for POL and Pol-α*, marked by diamonds) was plotted against the lagging:leading strand ratio determined using Okazaki-seq data (only ratios ≥ 1:1 for the latter are shown for clarity). There was high reproducibility between experiments in strand ratio preferences. Lines are lowess smoothed (see Methods) representations of the full datasets (representative examples given in f and g). f, g, Scatter plots illustrating the individual strand ratio data points for 2,001 nt windows, for stationary phase POL (f) and Pol-α* (g) yeast. Pearson’s cor=0.49, p < 2.2×10−16 for POL (f); cor=0.75, p< 2.2×10−16 for Pol-α* (g).
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Figure 9: Quantification of in vivo ribonucleotide incorporation by replicative polymerasesa, b, Representative alkaline gel electrophoresis of genomic DNA from yeast strains with mutant replicative DNA polymerases (a), with accompanying densitometry plots (b). Embedded ribonucleotides are detected by increased fragmentation of genomic DNA following alkaline treatment in an RNase H2-deficient (Δrnh201) background. Elevated rates are seen with all three mutant polymerases (indicated by *, as defined in Extended data Fig. 3a), and are reduced in Pol-ε′ which contains the point mutation M664L, a mutation that increases selectivity for dNTPs over rNTPs27. c, Quantification of average ribonucleotide incorporation in polymerase mutants from n=4 independent experiments. DNA isolated from mid-log phase cultures; error bars, SE. Overall ribonucleotide content is the product of incorporation frequency and the total contribution of each polymerase, resulting in the total ribonucleotide content detected to be highest for Pol-ε* (14,200 per genome), followed by Pol-δ* (4,300 per genome), Pol-α* (2,700 per genome), POL (1,900 per genome) and Pol-ε′ (860 per genome). d, The majority of the yeast genome exhibits directional asymmetry in replication (median 4:1 strand ratio). Count of genomic segments calculated for consecutive 2,001 nt windows over the yeast genome based on reanalysis of OF sequencing data17 denoted as ‘Okazaki-seq’. The strand asymmetry ratio was calculated after re-orienting all regions such that the predominant lagging strand was the forward strand. e-g, Genome-wide quantification of strand-specific incorporation of wild type and mutant replicative DNA polymerases determined by emRiboSeq reflects their roles in leading and lagging strand replication. A close to linear correlation with Okazaki-seq strand ratios is observed. The strand ratio preference for lagging strand ribonucleotide incorporation for independent libraries (including stationary phase libraries for POL and Pol-α*, marked by diamonds) was plotted against the lagging:leading strand ratio determined using Okazaki-seq data (only ratios ≥ 1:1 for the latter are shown for clarity). There was high reproducibility between experiments in strand ratio preferences. Lines are lowess smoothed (see Methods) representations of the full datasets (representative examples given in f and g). f, g, Scatter plots illustrating the individual strand ratio data points for 2,001 nt windows, for stationary phase POL (f) and Pol-α* (g) yeast. Pearson’s cor=0.49, p < 2.2×10−16 for POL (f); cor=0.75, p< 2.2×10−16 for Pol-α* (g).

Mentions: To provide biochemical validation, we performed alkaline gel electrophoresis on genomic DNA extracted from Pol-α L868M, Pol-δ L612M and Pol-ε M644G Δrnh201 yeast. Increased fragmentation was detected in all three strains (Extended data Fig. 4a-c) and elevated ribonucleotide incorporation was also detected in genomic DNA from stationary phase Pol-α L868M yeast (Fig. 4a-c), consistent with Pol-α tract retention in mature genomic DNA. To quantify the contribution of Pol-α DNA to the genome, we used densitometry measurements from the alkaline gels to calculate ribonucleotide incorporation rates28. We detected 1,500 embedded ribonucleotides per genome in Δrnh201 genomic DNA, which increased to 2,400 sites per genome for Pol-α L868M (Fig. 4c). Observed ribonucleotide incorporation rates correspond to the product of the incorporation frequency of each polymerase and the amount of DNA it contributes to the genome. Using the in vitro ribonucleotide incorporation rates of wildtype and mutant polymerases and the number of embedded ribonucleotides embedded in vivo (Extended data Fig. 3a and 4a-c) we estimated the relative contributions of each of the replicative polymerases to the genome (Fig. 4d), calculating the contribution of Pol-α to be 1.5 ± 0.3%.


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)

Quantification of in vivo ribonucleotide incorporation by replicative polymerasesa, b, Representative alkaline gel electrophoresis of genomic DNA from yeast strains with mutant replicative DNA polymerases (a), with accompanying densitometry plots (b). Embedded ribonucleotides are detected by increased fragmentation of genomic DNA following alkaline treatment in an RNase H2-deficient (Δrnh201) background. Elevated rates are seen with all three mutant polymerases (indicated by *, as defined in Extended data Fig. 3a), and are reduced in Pol-ε′ which contains the point mutation M664L, a mutation that increases selectivity for dNTPs over rNTPs27. c, Quantification of average ribonucleotide incorporation in polymerase mutants from n=4 independent experiments. DNA isolated from mid-log phase cultures; error bars, SE. Overall ribonucleotide content is the product of incorporation frequency and the total contribution of each polymerase, resulting in the total ribonucleotide content detected to be highest for Pol-ε* (14,200 per genome), followed by Pol-δ* (4,300 per genome), Pol-α* (2,700 per genome), POL (1,900 per genome) and Pol-ε′ (860 per genome). d, The majority of the yeast genome exhibits directional asymmetry in replication (median 4:1 strand ratio). Count of genomic segments calculated for consecutive 2,001 nt windows over the yeast genome based on reanalysis of OF sequencing data17 denoted as ‘Okazaki-seq’. The strand asymmetry ratio was calculated after re-orienting all regions such that the predominant lagging strand was the forward strand. e-g, Genome-wide quantification of strand-specific incorporation of wild type and mutant replicative DNA polymerases determined by emRiboSeq reflects their roles in leading and lagging strand replication. A close to linear correlation with Okazaki-seq strand ratios is observed. The strand ratio preference for lagging strand ribonucleotide incorporation for independent libraries (including stationary phase libraries for POL and Pol-α*, marked by diamonds) was plotted against the lagging:leading strand ratio determined using Okazaki-seq data (only ratios ≥ 1:1 for the latter are shown for clarity). There was high reproducibility between experiments in strand ratio preferences. Lines are lowess smoothed (see Methods) representations of the full datasets (representative examples given in f and g). f, g, Scatter plots illustrating the individual strand ratio data points for 2,001 nt windows, for stationary phase POL (f) and Pol-α* (g) yeast. Pearson’s cor=0.49, p < 2.2×10−16 for POL (f); cor=0.75, p< 2.2×10−16 for Pol-α* (g).
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Figure 9: Quantification of in vivo ribonucleotide incorporation by replicative polymerasesa, b, Representative alkaline gel electrophoresis of genomic DNA from yeast strains with mutant replicative DNA polymerases (a), with accompanying densitometry plots (b). Embedded ribonucleotides are detected by increased fragmentation of genomic DNA following alkaline treatment in an RNase H2-deficient (Δrnh201) background. Elevated rates are seen with all three mutant polymerases (indicated by *, as defined in Extended data Fig. 3a), and are reduced in Pol-ε′ which contains the point mutation M664L, a mutation that increases selectivity for dNTPs over rNTPs27. c, Quantification of average ribonucleotide incorporation in polymerase mutants from n=4 independent experiments. DNA isolated from mid-log phase cultures; error bars, SE. Overall ribonucleotide content is the product of incorporation frequency and the total contribution of each polymerase, resulting in the total ribonucleotide content detected to be highest for Pol-ε* (14,200 per genome), followed by Pol-δ* (4,300 per genome), Pol-α* (2,700 per genome), POL (1,900 per genome) and Pol-ε′ (860 per genome). d, The majority of the yeast genome exhibits directional asymmetry in replication (median 4:1 strand ratio). Count of genomic segments calculated for consecutive 2,001 nt windows over the yeast genome based on reanalysis of OF sequencing data17 denoted as ‘Okazaki-seq’. The strand asymmetry ratio was calculated after re-orienting all regions such that the predominant lagging strand was the forward strand. e-g, Genome-wide quantification of strand-specific incorporation of wild type and mutant replicative DNA polymerases determined by emRiboSeq reflects their roles in leading and lagging strand replication. A close to linear correlation with Okazaki-seq strand ratios is observed. The strand ratio preference for lagging strand ribonucleotide incorporation for independent libraries (including stationary phase libraries for POL and Pol-α*, marked by diamonds) was plotted against the lagging:leading strand ratio determined using Okazaki-seq data (only ratios ≥ 1:1 for the latter are shown for clarity). There was high reproducibility between experiments in strand ratio preferences. Lines are lowess smoothed (see Methods) representations of the full datasets (representative examples given in f and g). f, g, Scatter plots illustrating the individual strand ratio data points for 2,001 nt windows, for stationary phase POL (f) and Pol-α* (g) yeast. Pearson’s cor=0.49, p < 2.2×10−16 for POL (f); cor=0.75, p< 2.2×10−16 for Pol-α* (g).
Mentions: To provide biochemical validation, we performed alkaline gel electrophoresis on genomic DNA extracted from Pol-α L868M, Pol-δ L612M and Pol-ε M644G Δrnh201 yeast. Increased fragmentation was detected in all three strains (Extended data Fig. 4a-c) and elevated ribonucleotide incorporation was also detected in genomic DNA from stationary phase Pol-α L868M yeast (Fig. 4a-c), consistent with Pol-α tract retention in mature genomic DNA. To quantify the contribution of Pol-α DNA to the genome, we used densitometry measurements from the alkaline gels to calculate ribonucleotide incorporation rates28. We detected 1,500 embedded ribonucleotides per genome in Δrnh201 genomic DNA, which increased to 2,400 sites per genome for Pol-α L868M (Fig. 4c). Observed ribonucleotide incorporation rates correspond to the product of the incorporation frequency of each polymerase and the amount of DNA it contributes to the genome. Using the in vitro ribonucleotide incorporation rates of wildtype and mutant polymerases and the number of embedded ribonucleotides embedded in vivo (Extended data Fig. 3a and 4a-c) we estimated the relative contributions of each of the replicative polymerases to the genome (Fig. 4d), calculating the contribution of Pol-α to be 1.5 ± 0.3%.

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