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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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Related in: MedlinePlus

Pol-α synthesised DNA retention is independent of RNaseH2 processing of RNA primersa, b, The ribonucleotide content of genomic DNA is unchanged between Δrnh201 strains transformed with empty vector (−) or vector expressing Rnh201p separation of function mutant (sf), that retains the ability to cleave RNA:DNA hybrids, including RNA primers, but cannot cleave single embedded ribonucleotides39. In contrast, the same vector expressing wild type Rnh201p (wt) fully rescues alkaline sensitivity of the DNA. As complementation with the SOF mutant had no detectable effect on the ribonucleotide content seen in the Pol-α L868M Δrnh201 strain, retention of Pol-α synthesised DNA appears to be independent of a putative role for RNase H2 in RNA primer removal. c, Wild type and mutant Rnh201p are expressed at equal levels, as shown by immuno-detection of the C-terminal FLAG tag. Loading control, actin.
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Figure 10: Pol-α synthesised DNA retention is independent of RNaseH2 processing of RNA primersa, b, The ribonucleotide content of genomic DNA is unchanged between Δrnh201 strains transformed with empty vector (−) or vector expressing Rnh201p separation of function mutant (sf), that retains the ability to cleave RNA:DNA hybrids, including RNA primers, but cannot cleave single embedded ribonucleotides39. In contrast, the same vector expressing wild type Rnh201p (wt) fully rescues alkaline sensitivity of the DNA. As complementation with the SOF mutant had no detectable effect on the ribonucleotide content seen in the Pol-α L868M Δrnh201 strain, retention of Pol-α synthesised DNA appears to be independent of a putative role for RNase H2 in RNA primer removal. c, Wild type and mutant Rnh201p are expressed at equal levels, as shown by immuno-detection of the C-terminal FLAG tag. Loading control, actin.

Mentions: RNase H enzymes may contribute to removal of OF RNA primers16,38 and consequently Δrnh201 strains could have altered levels of Pol-α synthesised DNA to that seen in wild type strains. This confounding factor was excluded using an RNH201 separation of function (SOF) mutant39, which established that retention of Pol-α DNA was independent of a role for RNase H2 in RNA primer removal (Extended data Fig. 5).


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)

Pol-α synthesised DNA retention is independent of RNaseH2 processing of RNA primersa, b, The ribonucleotide content of genomic DNA is unchanged between Δrnh201 strains transformed with empty vector (−) or vector expressing Rnh201p separation of function mutant (sf), that retains the ability to cleave RNA:DNA hybrids, including RNA primers, but cannot cleave single embedded ribonucleotides39. In contrast, the same vector expressing wild type Rnh201p (wt) fully rescues alkaline sensitivity of the DNA. As complementation with the SOF mutant had no detectable effect on the ribonucleotide content seen in the Pol-α L868M Δrnh201 strain, retention of Pol-α synthesised DNA appears to be independent of a putative role for RNase H2 in RNA primer removal. c, Wild type and mutant Rnh201p are expressed at equal levels, as shown by immuno-detection of the C-terminal FLAG tag. Loading control, actin.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Pol-α synthesised DNA retention is independent of RNaseH2 processing of RNA primersa, b, The ribonucleotide content of genomic DNA is unchanged between Δrnh201 strains transformed with empty vector (−) or vector expressing Rnh201p separation of function mutant (sf), that retains the ability to cleave RNA:DNA hybrids, including RNA primers, but cannot cleave single embedded ribonucleotides39. In contrast, the same vector expressing wild type Rnh201p (wt) fully rescues alkaline sensitivity of the DNA. As complementation with the SOF mutant had no detectable effect on the ribonucleotide content seen in the Pol-α L868M Δrnh201 strain, retention of Pol-α synthesised DNA appears to be independent of a putative role for RNase H2 in RNA primer removal. c, Wild type and mutant Rnh201p are expressed at equal levels, as shown by immuno-detection of the C-terminal FLAG tag. Loading control, actin.
Mentions: RNase H enzymes may contribute to removal of OF RNA primers16,38 and consequently Δrnh201 strains could have altered levels of Pol-α synthesised DNA to that seen in wild type strains. This confounding factor was excluded using an RNH201 separation of function (SOF) mutant39, which established that retention of Pol-α DNA was independent of a role for RNase H2 in RNA primer removal (Extended data Fig. 5).

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