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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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EmRiboSeq methodology and validationa, Schematic of emRiboSeq library preparation. b-d, Validation of strand-specific detection of enzymatically generated nicks through linker-ligation. Nb.BtsI nicking endonuclease cleaves the bottom strand of its recognition site releasing a 5′ fragment (cyan) with a free 3′-OH group after denaturation, to which the sequencing adaptor (pink) is ligated, allowing sequencing and mapping of this site to the genome (b). Nb.BtsI libraries have high reproducibility between Δrnh201 POL and Δrnh201 Pol-α* (pol1-L868M) strains after normalising read counts to sequence tags per million (TPM). Bona fide Nb.BtsI sites were equally represented, at maximal frequency, in both libraries (c). Those with lower frequencies represented sites in close proximity to other Nb.BtsI sites, causing their partial loss during size selection. Additionally, Nb.BtsI-like sites were detected as the result of star activity. Libraries were also prepared using BciVI restriction enzyme digestion, that did not show such star activity (data not shown), allowing calculation of the site specificity for the method (>99.9%). Summed signal at Nb.BtsI sites shows >99.9% strand specificity (blue, correct strand; grey, opposite strand) and >99% single nucleotide resolution (d).
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Figure 7: EmRiboSeq methodology and validationa, Schematic of emRiboSeq library preparation. b-d, Validation of strand-specific detection of enzymatically generated nicks through linker-ligation. Nb.BtsI nicking endonuclease cleaves the bottom strand of its recognition site releasing a 5′ fragment (cyan) with a free 3′-OH group after denaturation, to which the sequencing adaptor (pink) is ligated, allowing sequencing and mapping of this site to the genome (b). Nb.BtsI libraries have high reproducibility between Δrnh201 POL and Δrnh201 Pol-α* (pol1-L868M) strains after normalising read counts to sequence tags per million (TPM). Bona fide Nb.BtsI sites were equally represented, at maximal frequency, in both libraries (c). Those with lower frequencies represented sites in close proximity to other Nb.BtsI sites, causing their partial loss during size selection. Additionally, Nb.BtsI-like sites were detected as the result of star activity. Libraries were also prepared using BciVI restriction enzyme digestion, that did not show such star activity (data not shown), allowing calculation of the site specificity for the method (>99.9%). Summed signal at Nb.BtsI sites shows >99.9% strand specificity (blue, correct strand; grey, opposite strand) and >99% single nucleotide resolution (d).

Mentions: To directly track the genome-wide contribution of polymerases we developed a next generation sequencing approach, which we term emRiboSeq (for embedded Ribonucleotide Sequencing), that determines the strand-specific, genome-wide distribution of embedded ribonucleotides. This is achieved by treatment of genomic DNA with recombinant type 2 ribonuclease H, to generate nicks 5′ of embedded ribonucleotides, followed by ligation of a sequencing adapter to the 3′-hydroxyl group of the deoxynucleotide immediately upstream of the ribonucleotide (Fig. 3b; Extended data Fig. 2a). Subsequent ion-semiconductor sequencing permits strand-specific mapping of ribonucleotide incorporation 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)

EmRiboSeq methodology and validationa, Schematic of emRiboSeq library preparation. b-d, Validation of strand-specific detection of enzymatically generated nicks through linker-ligation. Nb.BtsI nicking endonuclease cleaves the bottom strand of its recognition site releasing a 5′ fragment (cyan) with a free 3′-OH group after denaturation, to which the sequencing adaptor (pink) is ligated, allowing sequencing and mapping of this site to the genome (b). Nb.BtsI libraries have high reproducibility between Δrnh201 POL and Δrnh201 Pol-α* (pol1-L868M) strains after normalising read counts to sequence tags per million (TPM). Bona fide Nb.BtsI sites were equally represented, at maximal frequency, in both libraries (c). Those with lower frequencies represented sites in close proximity to other Nb.BtsI sites, causing their partial loss during size selection. Additionally, Nb.BtsI-like sites were detected as the result of star activity. Libraries were also prepared using BciVI restriction enzyme digestion, that did not show such star activity (data not shown), allowing calculation of the site specificity for the method (>99.9%). Summed signal at Nb.BtsI sites shows >99.9% strand specificity (blue, correct strand; grey, opposite strand) and >99% single nucleotide resolution (d).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374164&req=5

Figure 7: EmRiboSeq methodology and validationa, Schematic of emRiboSeq library preparation. b-d, Validation of strand-specific detection of enzymatically generated nicks through linker-ligation. Nb.BtsI nicking endonuclease cleaves the bottom strand of its recognition site releasing a 5′ fragment (cyan) with a free 3′-OH group after denaturation, to which the sequencing adaptor (pink) is ligated, allowing sequencing and mapping of this site to the genome (b). Nb.BtsI libraries have high reproducibility between Δrnh201 POL and Δrnh201 Pol-α* (pol1-L868M) strains after normalising read counts to sequence tags per million (TPM). Bona fide Nb.BtsI sites were equally represented, at maximal frequency, in both libraries (c). Those with lower frequencies represented sites in close proximity to other Nb.BtsI sites, causing their partial loss during size selection. Additionally, Nb.BtsI-like sites were detected as the result of star activity. Libraries were also prepared using BciVI restriction enzyme digestion, that did not show such star activity (data not shown), allowing calculation of the site specificity for the method (>99.9%). Summed signal at Nb.BtsI sites shows >99.9% strand specificity (blue, correct strand; grey, opposite strand) and >99% single nucleotide resolution (d).
Mentions: To directly track the genome-wide contribution of polymerases we developed a next generation sequencing approach, which we term emRiboSeq (for embedded Ribonucleotide Sequencing), that determines the strand-specific, genome-wide distribution of embedded ribonucleotides. This is achieved by treatment of genomic DNA with recombinant type 2 ribonuclease H, to generate nicks 5′ of embedded ribonucleotides, followed by ligation of a sequencing adapter to the 3′-hydroxyl group of the deoxynucleotide immediately upstream of the ribonucleotide (Fig. 3b; Extended data Fig. 2a). Subsequent ion-semiconductor sequencing permits strand-specific mapping of ribonucleotide incorporation 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