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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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Pol-α DNA synthesis contributes ~1.5% of the mature genomea, b Increased ribonucleotide incorporation in Pol-α* stationary phase yeast is detected by alkaline gel electrophoresis. c, Quantification confirms significantly elevated rates (n=6; error bars, SE; paired two-sided t-test) in the Pol-α* genome. d, Estimate of relative contribution of polymerases to the genome (n=4; error bars, SE).
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Figure 4: Pol-α DNA synthesis contributes ~1.5% of the mature genomea, b Increased ribonucleotide incorporation in Pol-α* stationary phase yeast is detected by alkaline gel electrophoresis. c, Quantification confirms significantly elevated rates (n=6; error bars, SE; paired two-sided t-test) in the Pol-α* genome. d, Estimate of relative contribution of polymerases to the genome (n=4; error bars, SE).

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)

Pol-α DNA synthesis contributes ~1.5% of the mature genomea, b Increased ribonucleotide incorporation in Pol-α* stationary phase yeast is detected by alkaline gel electrophoresis. c, Quantification confirms significantly elevated rates (n=6; error bars, SE; paired two-sided t-test) in the Pol-α* genome. d, Estimate of relative contribution of polymerases to the genome (n=4; error bars, SE).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374164&req=5

Figure 4: Pol-α DNA synthesis contributes ~1.5% of the mature genomea, b Increased ribonucleotide incorporation in Pol-α* stationary phase yeast is detected by alkaline gel electrophoresis. c, Quantification confirms significantly elevated rates (n=6; error bars, SE; paired two-sided t-test) in the Pol-α* genome. d, Estimate of relative contribution of polymerases to the genome (n=4; error bars, SE).
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