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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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OF mutational signatures are conserved in humansa, Nucleotide substitutions (plotted as GERP scores) are elevated immediately adjacent to TF NFYA binding sites. Pink to brown: lower to higher quartiles of ChIP-seq peak height (reflecting strength of binding/occupancy). Stronger binding correlates with substitution rate in the ‘shoulder’ region (*). b, Elevated substitution rates are not a consequence of local sequence composition effects. Strongest binding sites (brown) compared to 3-mer preserving shuffle (black). c, Model: Nucleotide substitution profiles are the sum of mutation rate and selective pressure. d, Interspecies substitution rates are also elevated adjacent to DNase I footprint edges (*). Sequences aligned to left footprint edges as indicated in schematic. Right footprint edge is indistinct due to heterogeneity in footprint length. Substitution rates are no longer increased after 3-mer preserving shuffle from local flanking sequences (black). 95% confidence intervals, brown dashes and grey shading (b, d).
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Figure 5: OF mutational signatures are conserved in humansa, Nucleotide substitutions (plotted as GERP scores) are elevated immediately adjacent to TF NFYA binding sites. Pink to brown: lower to higher quartiles of ChIP-seq peak height (reflecting strength of binding/occupancy). Stronger binding correlates with substitution rate in the ‘shoulder’ region (*). b, Elevated substitution rates are not a consequence of local sequence composition effects. Strongest binding sites (brown) compared to 3-mer preserving shuffle (black). c, Model: Nucleotide substitution profiles are the sum of mutation rate and selective pressure. d, Interspecies substitution rates are also elevated adjacent to DNase I footprint edges (*). Sequences aligned to left footprint edges as indicated in schematic. Right footprint edge is indistinct due to heterogeneity in footprint length. Substitution rates are no longer increased after 3-mer preserving shuffle from local flanking sequences (black). 95% confidence intervals, brown dashes and grey shading (b, d).

Mentions: As OF processing is a conserved process in eukaryotes, we next considered whether an OF-related mutational signature was also present in humans. Substitution rates are also elevated at nucleosome cores in humans7 with an identical distribution to yeast. Furthermore, the TF NFYA has an unexplained “shoulder” of elevated substitution proximal to its binding sites40, reminiscent of the Reb1 pattern (Fig. 1b). We therefore investigated if similar mutational patterns are present at other experimentally defined human TF and chromatin protein binding sites. Elevated inter-species nucleotide substitution rates were detected flanking essential binding site residues, for many, but not all TFs, as well as CTCF binding sites (Fig. 5a,b and Extended data Fig. 6). Substitution rates were measured using GERP scores, which quantify nucleotide substitution rates relative to a genome wide expectation of neutral evolution41, such that a negative GERP score indicates increased nucleotide substitution rates. Furthermore, elevation in mutation rate correlated with the degree of enrichment reported in exoChIP datasets for these proteins, likely reflecting the strength of binding or frequency of occupancy at specific sites, which would be expected to influence pol-δ processivity and consequent mutation levels.


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)

OF mutational signatures are conserved in humansa, Nucleotide substitutions (plotted as GERP scores) are elevated immediately adjacent to TF NFYA binding sites. Pink to brown: lower to higher quartiles of ChIP-seq peak height (reflecting strength of binding/occupancy). Stronger binding correlates with substitution rate in the ‘shoulder’ region (*). b, Elevated substitution rates are not a consequence of local sequence composition effects. Strongest binding sites (brown) compared to 3-mer preserving shuffle (black). c, Model: Nucleotide substitution profiles are the sum of mutation rate and selective pressure. d, Interspecies substitution rates are also elevated adjacent to DNase I footprint edges (*). Sequences aligned to left footprint edges as indicated in schematic. Right footprint edge is indistinct due to heterogeneity in footprint length. Substitution rates are no longer increased after 3-mer preserving shuffle from local flanking sequences (black). 95% confidence intervals, brown dashes and grey shading (b, d).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374164&req=5

Figure 5: OF mutational signatures are conserved in humansa, Nucleotide substitutions (plotted as GERP scores) are elevated immediately adjacent to TF NFYA binding sites. Pink to brown: lower to higher quartiles of ChIP-seq peak height (reflecting strength of binding/occupancy). Stronger binding correlates with substitution rate in the ‘shoulder’ region (*). b, Elevated substitution rates are not a consequence of local sequence composition effects. Strongest binding sites (brown) compared to 3-mer preserving shuffle (black). c, Model: Nucleotide substitution profiles are the sum of mutation rate and selective pressure. d, Interspecies substitution rates are also elevated adjacent to DNase I footprint edges (*). Sequences aligned to left footprint edges as indicated in schematic. Right footprint edge is indistinct due to heterogeneity in footprint length. Substitution rates are no longer increased after 3-mer preserving shuffle from local flanking sequences (black). 95% confidence intervals, brown dashes and grey shading (b, d).
Mentions: As OF processing is a conserved process in eukaryotes, we next considered whether an OF-related mutational signature was also present in humans. Substitution rates are also elevated at nucleosome cores in humans7 with an identical distribution to yeast. Furthermore, the TF NFYA has an unexplained “shoulder” of elevated substitution proximal to its binding sites40, reminiscent of the Reb1 pattern (Fig. 1b). We therefore investigated if similar mutational patterns are present at other experimentally defined human TF and chromatin protein binding sites. Elevated inter-species nucleotide substitution rates were detected flanking essential binding site residues, for many, but not all TFs, as well as CTCF binding sites (Fig. 5a,b and Extended data Fig. 6). Substitution rates were measured using GERP scores, which quantify nucleotide substitution rates relative to a genome wide expectation of neutral evolution41, such that a negative GERP score indicates increased nucleotide substitution rates. Furthermore, elevation in mutation rate correlated with the degree of enrichment reported in exoChIP datasets for these proteins, likely reflecting the strength of binding or frequency of occupancy at specific sites, which would be expected to influence pol-δ processivity and consequent mutation levels.

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