Limits...
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.

Show MeSH

Related in: MedlinePlus

Mapping DNA synthesis in vivo using emRiboSeqa, Replicative polymerases can be tracked using point mutants with elevated ribonucleotide incorporation. Schematic of replication fork with Pol-ε (*, M644G mutant) and ribonucleotide incorporation rates for each polymerase. Embedded ribonucleotides (R) highlighted. b, Schematic of emRiboSeq methodology. c, Schematic of replication. d, e, Mapping of leading/lagging strand synthesis and replication origins using emRiboSeq. Ratio of OFs reads17 between forward and reverse strands of chromosome 10 (d) corresponds to the ratio of their respective ribonucleotide content (e) for Pol-δ* (orange), whereas Pol-ε* shows negative correlation (cyan). Intersections with x-axis correspond to replication origins and termination regions (c-e). Experimentally validated origins (dotted pink lines). f, Pol-α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand. Strand ratios are shown as best fit splines, y-axes log2 of ratios (d-f)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4374164&req=5

Figure 3: Mapping DNA synthesis in vivo using emRiboSeqa, Replicative polymerases can be tracked using point mutants with elevated ribonucleotide incorporation. Schematic of replication fork with Pol-ε (*, M644G mutant) and ribonucleotide incorporation rates for each polymerase. Embedded ribonucleotides (R) highlighted. b, Schematic of emRiboSeq methodology. c, Schematic of replication. d, e, Mapping of leading/lagging strand synthesis and replication origins using emRiboSeq. Ratio of OFs reads17 between forward and reverse strands of chromosome 10 (d) corresponds to the ratio of their respective ribonucleotide content (e) for Pol-δ* (orange), whereas Pol-ε* shows negative correlation (cyan). Intersections with x-axis correspond to replication origins and termination regions (c-e). Experimentally validated origins (dotted pink lines). f, Pol-α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand. Strand ratios are shown as best fit splines, y-axes log2 of ratios (d-f)

Mentions: To address where error-prone Pol-α DNA is retained in vivo, we utilised the incorporation of ribonucleotides into genomic DNA to track the activity of specific DNA polymerases. Ribonucleotides are covalently incorporated into genomic DNA by replicative polymerases27,28, although they are normally efficiently removed by Ribonucleotide Excision Repair (RER), a process initiated by the RNase H2 enzyme29. In RNase H2 deficient budding yeast such ribonucleotides are generally well tolerated: Δrnh201 yeast has proliferation rates identical to wild type under normal growth conditions27, and therefore in this genetic background ribonucleotides can be used as a ‘label’ to track polymerase activity. Furthermore, the contribution of specific polymerases can be studied using polymerases with catalytic site point mutations (Pol-α L868M, Pol-δ L612M and Pol-ε M644G) that incorporate ribonucleotides at higher rates than their wildtype counterparts (21,26,27,30 and JS Williams, AR Clausen & TA Kunkel, personal communication; Fig. 3a). Yeast strains expressing these mutant polymerases have previously been used to demonstrate that Pol-ε and Pol-δ are the major leading and lagging strand polymerases respectively, by measuring strand-specific alkaline sensitivity of particular genomic loci30-32.


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)

Mapping DNA synthesis in vivo using emRiboSeqa, Replicative polymerases can be tracked using point mutants with elevated ribonucleotide incorporation. Schematic of replication fork with Pol-ε (*, M644G mutant) and ribonucleotide incorporation rates for each polymerase. Embedded ribonucleotides (R) highlighted. b, Schematic of emRiboSeq methodology. c, Schematic of replication. d, e, Mapping of leading/lagging strand synthesis and replication origins using emRiboSeq. Ratio of OFs reads17 between forward and reverse strands of chromosome 10 (d) corresponds to the ratio of their respective ribonucleotide content (e) for Pol-δ* (orange), whereas Pol-ε* shows negative correlation (cyan). Intersections with x-axis correspond to replication origins and termination regions (c-e). Experimentally validated origins (dotted pink lines). f, Pol-α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand. Strand ratios are shown as best fit splines, y-axes log2 of ratios (d-f)
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Mapping DNA synthesis in vivo using emRiboSeqa, Replicative polymerases can be tracked using point mutants with elevated ribonucleotide incorporation. Schematic of replication fork with Pol-ε (*, M644G mutant) and ribonucleotide incorporation rates for each polymerase. Embedded ribonucleotides (R) highlighted. b, Schematic of emRiboSeq methodology. c, Schematic of replication. d, e, Mapping of leading/lagging strand synthesis and replication origins using emRiboSeq. Ratio of OFs reads17 between forward and reverse strands of chromosome 10 (d) corresponds to the ratio of their respective ribonucleotide content (e) for Pol-δ* (orange), whereas Pol-ε* shows negative correlation (cyan). Intersections with x-axis correspond to replication origins and termination regions (c-e). Experimentally validated origins (dotted pink lines). f, Pol-α* DNA is detected genome-wide by emRiboSeq as a component of the lagging strand. Strand ratios are shown as best fit splines, y-axes log2 of ratios (d-f)
Mentions: To address where error-prone Pol-α DNA is retained in vivo, we utilised the incorporation of ribonucleotides into genomic DNA to track the activity of specific DNA polymerases. Ribonucleotides are covalently incorporated into genomic DNA by replicative polymerases27,28, although they are normally efficiently removed by Ribonucleotide Excision Repair (RER), a process initiated by the RNase H2 enzyme29. In RNase H2 deficient budding yeast such ribonucleotides are generally well tolerated: Δrnh201 yeast has proliferation rates identical to wild type under normal growth conditions27, and therefore in this genetic background ribonucleotides can be used as a ‘label’ to track polymerase activity. Furthermore, the contribution of specific polymerases can be studied using polymerases with catalytic site point mutations (Pol-α L868M, Pol-δ L612M and Pol-ε M644G) that incorporate ribonucleotides at higher rates than their wildtype counterparts (21,26,27,30 and JS Williams, AR Clausen & TA Kunkel, personal communication; Fig. 3a). Yeast strains expressing these mutant polymerases have previously been used to demonstrate that Pol-ε and Pol-δ are the major leading and lagging strand polymerases respectively, by measuring strand-specific alkaline sensitivity of particular genomic loci30-32.

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