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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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Model: Pol-α DNA tract retention downstream of protein binding sitesa, OF priming occurs stochastically, with the 5′ end of each OF initially synthesised by Pol-α and the remainder of the OF synthesised by Pol-δ. b, c, OF processing: when Pol-δ encounters the previously synthesised OF, Pol-δ continues to synthesise DNA displacing the 5′ end of the downstream OF, which is removed by nucleases to result in mature OFs which are then ligated. The OJs of such mature OFs prior to ligation were detected by Smith and Whitehouse17 after depletion of temperature sensitive DNA ligase I. They demonstrated that if a protein barrier is encountered (grey circle) Pol-δ progression is impaired, leading to reduced removal of the downstream OF (b). Given that ~1.5% of the mature genome is synthesised by Pol-α, a proportion of lagging strands will retain Pol-α synthesised DNA (red). When Pol-δ progression is impaired by protein binding, this will lead to an increased fraction of fragments containing Pol-α synthesised DNA downstream of such sites (c).
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Figure 13: Model: Pol-α DNA tract retention downstream of protein binding sitesa, OF priming occurs stochastically, with the 5′ end of each OF initially synthesised by Pol-α and the remainder of the OF synthesised by Pol-δ. b, c, OF processing: when Pol-δ encounters the previously synthesised OF, Pol-δ continues to synthesise DNA displacing the 5′ end of the downstream OF, which is removed by nucleases to result in mature OFs which are then ligated. The OJs of such mature OFs prior to ligation were detected by Smith and Whitehouse17 after depletion of temperature sensitive DNA ligase I. They demonstrated that if a protein barrier is encountered (grey circle) Pol-δ progression is impaired, leading to reduced removal of the downstream OF (b). Given that ~1.5% of the mature genome is synthesised by Pol-α, a proportion of lagging strands will retain Pol-α synthesised DNA (red). When Pol-δ progression is impaired by protein binding, this will lead to an increased fraction of fragments containing Pol-α synthesised DNA downstream of such sites (c).

Mentions: A direct relationship between OF junctions and mutation frequency is indicated by the significant correlations between substitution rate and OF junction sites at diverse protein binding sites, although future experimental validation will be needed to formally establish causality. We find that substitution rates are specifically elevated downstream of such junction sites, suggesting a replicative origin for such mutations. As Pol-α DNA tracts occur genome-wide, and Pol-δ processing of OFs is impaired by DNA-bound proteins17, we propose that retention of Pol-α DNA is increased at these functionally important sites, and is responsible for the increased mutation rate (Fig. 2c). Replication fidelity processes, including efficient MMR at the 5′ end of OFs25,45, will mitigate Pol-α replication errors. Additionally, Pol-α DNA will be incorporated at relatively low frequency (Extended data Fig. 8), with the majority of DNA at such sites still synthesised by Pol-δ and Pol-ε. However, over evolutionary timescales, it appears that these processes are insufficient to fully compensate for the lack of Pol-α proofreading activity. An alternative possibility is that protein binding may impair access of replication-related repair factors, such as Exo1 to correct errors in Pol-α synthesised DNA45. However, it does not appear that the MMR machinery is generally obstructed at such sites, as MMR efficiency at nucleosomes is reported to be uniform with respect to dyad position24.


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)

Model: Pol-α DNA tract retention downstream of protein binding sitesa, OF priming occurs stochastically, with the 5′ end of each OF initially synthesised by Pol-α and the remainder of the OF synthesised by Pol-δ. b, c, OF processing: when Pol-δ encounters the previously synthesised OF, Pol-δ continues to synthesise DNA displacing the 5′ end of the downstream OF, which is removed by nucleases to result in mature OFs which are then ligated. The OJs of such mature OFs prior to ligation were detected by Smith and Whitehouse17 after depletion of temperature sensitive DNA ligase I. They demonstrated that if a protein barrier is encountered (grey circle) Pol-δ progression is impaired, leading to reduced removal of the downstream OF (b). Given that ~1.5% of the mature genome is synthesised by Pol-α, a proportion of lagging strands will retain Pol-α synthesised DNA (red). When Pol-δ progression is impaired by protein binding, this will lead to an increased fraction of fragments containing Pol-α synthesised DNA downstream of such sites (c).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4374164&req=5

Figure 13: Model: Pol-α DNA tract retention downstream of protein binding sitesa, OF priming occurs stochastically, with the 5′ end of each OF initially synthesised by Pol-α and the remainder of the OF synthesised by Pol-δ. b, c, OF processing: when Pol-δ encounters the previously synthesised OF, Pol-δ continues to synthesise DNA displacing the 5′ end of the downstream OF, which is removed by nucleases to result in mature OFs which are then ligated. The OJs of such mature OFs prior to ligation were detected by Smith and Whitehouse17 after depletion of temperature sensitive DNA ligase I. They demonstrated that if a protein barrier is encountered (grey circle) Pol-δ progression is impaired, leading to reduced removal of the downstream OF (b). Given that ~1.5% of the mature genome is synthesised by Pol-α, a proportion of lagging strands will retain Pol-α synthesised DNA (red). When Pol-δ progression is impaired by protein binding, this will lead to an increased fraction of fragments containing Pol-α synthesised DNA downstream of such sites (c).
Mentions: A direct relationship between OF junctions and mutation frequency is indicated by the significant correlations between substitution rate and OF junction sites at diverse protein binding sites, although future experimental validation will be needed to formally establish causality. We find that substitution rates are specifically elevated downstream of such junction sites, suggesting a replicative origin for such mutations. As Pol-α DNA tracts occur genome-wide, and Pol-δ processing of OFs is impaired by DNA-bound proteins17, we propose that retention of Pol-α DNA is increased at these functionally important sites, and is responsible for the increased mutation rate (Fig. 2c). Replication fidelity processes, including efficient MMR at the 5′ end of OFs25,45, will mitigate Pol-α replication errors. Additionally, Pol-α DNA will be incorporated at relatively low frequency (Extended data Fig. 8), with the majority of DNA at such sites still synthesised by Pol-δ and Pol-ε. However, over evolutionary timescales, it appears that these processes are insufficient to fully compensate for the lack of Pol-α proofreading activity. An alternative possibility is that protein binding may impair access of replication-related repair factors, such as Exo1 to correct errors in Pol-α synthesised DNA45. However, it does not appear that the MMR machinery is generally obstructed at such sites, as MMR efficiency at nucleosomes is reported to be uniform with respect to dyad position24.

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