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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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Increased OJ and polymorphism rates correlate at binding sites of different nucleosome classes and at Rap1 binding sitesa-f, OJ and polymorphism rates are strongly correlated for different classes of nucleosomes. Data presented as in Fig. 1a, for different sub-classes of S. cerevisiae nucleosomes, demonstrating that OJ and polymorphism rates co-vary in all cases. Transcription start site (TSS) proximal nucleosomes (d) are likely subject to strong and asymmetrically distributed selective constraints, which likely explains the modestly reduced correlation for this subset. Such TSS proximal nucleosomes were excluded from analyses of other categories presented (b, c, e, f), except ‘All nucleosomes’ (a). g, OJ and polymorphism rates are correlated for the S. cerevisiae TF, Rap1. Data presented, as for Reb1 in Fig. 1b, show elevated OJ and polymorphism rates around its binding site, with a dip corresponding to its central recognition sequence. h-j, Elevated polymorphism and OJ rates at Rap1 (h), nucleosome (i) and Reb1 binding sites (j) are not due to biases in nucleotide content. Distributions calculated as for g, Fig. 1a and b respectively, using a 3-mer preserving genome shuffle. Pink shaded areas, 95% confidence intervals for nucleotide substitution rates (100 shuffles). k, l, Polymorphism (red) and between-species (black) substitution rates are highly correlated for nucleosome (k) and Reb1 (l) binding sites. Best fit splines shown only. Y-axes scaled to demonstrate similar shape distribution. Values plotted as percentage relative to the mean rate for all data points (central 11 nt excluded for calculation of mean in l).
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Figure 6: Increased OJ and polymorphism rates correlate at binding sites of different nucleosome classes and at Rap1 binding sitesa-f, OJ and polymorphism rates are strongly correlated for different classes of nucleosomes. Data presented as in Fig. 1a, for different sub-classes of S. cerevisiae nucleosomes, demonstrating that OJ and polymorphism rates co-vary in all cases. Transcription start site (TSS) proximal nucleosomes (d) are likely subject to strong and asymmetrically distributed selective constraints, which likely explains the modestly reduced correlation for this subset. Such TSS proximal nucleosomes were excluded from analyses of other categories presented (b, c, e, f), except ‘All nucleosomes’ (a). g, OJ and polymorphism rates are correlated for the S. cerevisiae TF, Rap1. Data presented, as for Reb1 in Fig. 1b, show elevated OJ and polymorphism rates around its binding site, with a dip corresponding to its central recognition sequence. h-j, Elevated polymorphism and OJ rates at Rap1 (h), nucleosome (i) and Reb1 binding sites (j) are not due to biases in nucleotide content. Distributions calculated as for g, Fig. 1a and b respectively, using a 3-mer preserving genome shuffle. Pink shaded areas, 95% confidence intervals for nucleotide substitution rates (100 shuffles). k, l, Polymorphism (red) and between-species (black) substitution rates are highly correlated for nucleosome (k) and Reb1 (l) binding sites. Best fit splines shown only. Y-axes scaled to demonstrate similar shape distribution. Values plotted as percentage relative to the mean rate for all data points (central 11 nt excluded for calculation of mean in l).

Mentions: We were struck by the similarity of the distribution of S. cerevisiae OJ sites at nucleosomes17 to that previously reported for nucleotide substitutions7,8,10-12, and set out to investigate the potential reasons for this. We established that nucleotide substitution and OJ distributions are highly correlated (Pearson’s correlation coefficient = 0.76, p = 2.2·10−16) and essentially identical in pattern (Fig. 1a). Furthermore, differences in OJ distribution by nucleosome type (genic vs non-genic), spacing or consistency of binding were mirrored by the substitution rate distribution (Extended data Fig. 1a-f). We found similar strong correlation in the regions directly surrounding TF binding sites of Reb1 (Fig. 1b; Pearson’s cor = 0.57, p = 5.6·10−15), and Rap1 (Extended data Fig. 1g), providing further evidence for a direct association. At the sequence-specific binding sites themselves, substitution rates were depressed relative to the OJ, resulting from strong selection pressure to maintain TF binding, and obscuring any mutational signal at these nucleotides.


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)

Increased OJ and polymorphism rates correlate at binding sites of different nucleosome classes and at Rap1 binding sitesa-f, OJ and polymorphism rates are strongly correlated for different classes of nucleosomes. Data presented as in Fig. 1a, for different sub-classes of S. cerevisiae nucleosomes, demonstrating that OJ and polymorphism rates co-vary in all cases. Transcription start site (TSS) proximal nucleosomes (d) are likely subject to strong and asymmetrically distributed selective constraints, which likely explains the modestly reduced correlation for this subset. Such TSS proximal nucleosomes were excluded from analyses of other categories presented (b, c, e, f), except ‘All nucleosomes’ (a). g, OJ and polymorphism rates are correlated for the S. cerevisiae TF, Rap1. Data presented, as for Reb1 in Fig. 1b, show elevated OJ and polymorphism rates around its binding site, with a dip corresponding to its central recognition sequence. h-j, Elevated polymorphism and OJ rates at Rap1 (h), nucleosome (i) and Reb1 binding sites (j) are not due to biases in nucleotide content. Distributions calculated as for g, Fig. 1a and b respectively, using a 3-mer preserving genome shuffle. Pink shaded areas, 95% confidence intervals for nucleotide substitution rates (100 shuffles). k, l, Polymorphism (red) and between-species (black) substitution rates are highly correlated for nucleosome (k) and Reb1 (l) binding sites. Best fit splines shown only. Y-axes scaled to demonstrate similar shape distribution. Values plotted as percentage relative to the mean rate for all data points (central 11 nt excluded for calculation of mean in l).
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Figure 6: Increased OJ and polymorphism rates correlate at binding sites of different nucleosome classes and at Rap1 binding sitesa-f, OJ and polymorphism rates are strongly correlated for different classes of nucleosomes. Data presented as in Fig. 1a, for different sub-classes of S. cerevisiae nucleosomes, demonstrating that OJ and polymorphism rates co-vary in all cases. Transcription start site (TSS) proximal nucleosomes (d) are likely subject to strong and asymmetrically distributed selective constraints, which likely explains the modestly reduced correlation for this subset. Such TSS proximal nucleosomes were excluded from analyses of other categories presented (b, c, e, f), except ‘All nucleosomes’ (a). g, OJ and polymorphism rates are correlated for the S. cerevisiae TF, Rap1. Data presented, as for Reb1 in Fig. 1b, show elevated OJ and polymorphism rates around its binding site, with a dip corresponding to its central recognition sequence. h-j, Elevated polymorphism and OJ rates at Rap1 (h), nucleosome (i) and Reb1 binding sites (j) are not due to biases in nucleotide content. Distributions calculated as for g, Fig. 1a and b respectively, using a 3-mer preserving genome shuffle. Pink shaded areas, 95% confidence intervals for nucleotide substitution rates (100 shuffles). k, l, Polymorphism (red) and between-species (black) substitution rates are highly correlated for nucleosome (k) and Reb1 (l) binding sites. Best fit splines shown only. Y-axes scaled to demonstrate similar shape distribution. Values plotted as percentage relative to the mean rate for all data points (central 11 nt excluded for calculation of mean in l).
Mentions: We were struck by the similarity of the distribution of S. cerevisiae OJ sites at nucleosomes17 to that previously reported for nucleotide substitutions7,8,10-12, and set out to investigate the potential reasons for this. We established that nucleotide substitution and OJ distributions are highly correlated (Pearson’s correlation coefficient = 0.76, p = 2.2·10−16) and essentially identical in pattern (Fig. 1a). Furthermore, differences in OJ distribution by nucleosome type (genic vs non-genic), spacing or consistency of binding were mirrored by the substitution rate distribution (Extended data Fig. 1a-f). We found similar strong correlation in the regions directly surrounding TF binding sites of Reb1 (Fig. 1b; Pearson’s cor = 0.57, p = 5.6·10−15), and Rap1 (Extended data Fig. 1g), providing further evidence for a direct association. At the sequence-specific binding sites themselves, substitution rates were depressed relative to the OJ, resulting from strong selection pressure to maintain TF binding, and obscuring any mutational signal at these nucleotides.

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