Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissuespecific tumors. To examine relationships between such heterogeneity and variations in leading-and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.
Ribonucleotides are frequently incorporated into DNA during eukaryotic replication. Here we map the genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5′-DNA end-mapping method, Hydrolytic End Sequencing. HydEn-Seq of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the role of DNA polymerases α and δ in lagging strand replication and of DNA polymerase ε in leading strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-Seq also reveals strand-specific 5′-DNA ends at mitochondrial replication origins, suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-Seq can be used to track replication enzymology in other organisms.
To maintain genome stability, mismatch repair of nuclear DNA replication errors must be directed to the nascent strand, likely by DNA ends and PCNA. Here we show that the efficiency of mismatch repair in Saccharomyces cerevisiae is reduced by inactivating RNase H2, which nicks DNA containing ribonucleotides incorporated during replication. In strains encoding mutator polymerases, this reduction is preferential for repair of mismatches made by leading strand DNA polymerase ε as compared to lagging strand DNA polymerase δ. The results suggest that RNase H2-dependent processing of ribonucleotides transiently present in DNA after replication may direct mismatch repair to the continuously replicated nascent leading strand.
In both budding and fission yeast, a large number of ribonucleotides are incorporated into DNA during replication by the major replicative polymerases (Pols α, δ, and ε). They are subsequently removed by RNase H2-dependent repair, which if defective leads to replication stress and genome instability. To extend these studies to humans, where an RNase H2 defect results in an autoimmune disease, here we compare the ability of human and yeast Pol δ to incorporate, proofread, and bypass ribonucleotides during DNA synthesis. In reactions containing nucleotide concentrations estimated to be present in mammalian cells, human Pol δ stably incorporates one rNTP for approximately 2,000 dNTPs, a ratio similar to that for yeast Pol δ. This result predicts that human Pol δ may introduce more than a million ribonucleotides into the nuclear genome per replication cycle, an amount recently reported to be present in the genome of RNase H2-defective mouse cells. Consistent with such abundant stable incorporation, we show that the 3´-exonuclease activity of yeast and human Pol δ largely fails to edit ribonucleotides during polymerization. We also show that, like yeast Pol δ, human Pol δ pauses as it bypasses ribonucleotides in DNA templates, with four consecutive ribonucleotides in a DNA template being more problematic than single ribonucleotides. In conjunction with recent studies in yeast and mice, this ribonucleotide incorporation may be relevant to impaired development and disease when RNase H2 is defective in mammals. As one tool to investigate ribonucleotide incorporation by Pol δ in human cells, we show that human Pol δ containing a Leu606Met substitution in the polymerase active site incorporates 7-fold more ribonucleotides into DNA than does wild type Pol δ.
Sequencing of whole cancer genomes has revealed an abundance of recurrent mutations in gene-regulatory promoter regions, in particular in melanoma where strong mutation hotspots are observed adjacent to ETS-family transcription factor (TF) binding sites. While sometimes interpreted as functional driver events, these mutations are commonly believed to be due to locally inhibited DNA repair. Here, we first show that low-dose UV light induces mutations preferably at a known ETS promoter hotspot in cultured cells even in the absence of global or transcription-coupled nucleotide excision repair (NER). Further, by genome-wide mapping of cyclobutane pyrimidine dimers (CPDs) shortly after UV exposure and thus before DNA repair, we find that ETS-related mutation hotspots exhibit strong increases in CPD formation efficacy in a manner consistent with tumor mutation data at the single-base level. Analysis of a large whole genome cohort illustrates the widespread contribution of this effect to recurrent mutations in melanoma. While inhibited NER underlies a general increase in somatic mutation burden in regulatory elements including ETS sites, our data supports that elevated DNA damage formation at specific genomic bases is at the core of the prominent promoter mutation hotspots seen in skin cancers, thus explaining a key phenomenon in whole-genome cancer analyses.
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