Division of Labor at the Eukaryotic Replication Fork

Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA.
Molecular cell (Impact Factor: 14.02). 05/2008; 30(2):137-44. DOI: 10.1016/j.molcel.2008.02.022
Source: PubMed


DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon) are both required for efficient replication of the nuclear genome, yet the division of labor between these enzymes has remained unclear for many years. Here we investigate the contribution of Pol delta to replication of the leading and lagging strand templates in Saccharomyces cerevisiae using a mutant Pol delta allele (pol3-L612M) whose error rate is higher for one mismatch (e.g., T x dGTP) than for its complement (A x dCTP). We find that strand-specific mutation rates strongly depend on the orientation of a reporter gene relative to an adjacent replication origin, in a manner implying that >90% of Pol delta replication is performed using the lagging strand template. When combined with recent evidence implicating Pol epsilon in leading strand replication, these data support a model of the replication fork wherein the leading and lagging strand templates are primarily copied by Pol epsilon and Pol delta, respectively.

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Available from: Carrie M Stith, Mar 13, 2015
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    • "The S. cerevisiae strains used here are isogenic derivatives of strain |(−2)|-7B-YUNI300 (MATa CAN1 his7-2 leu2-::kanMX ura3-trp1-289 ade2-1 lys2-GG2899-2900 agp1::URA3-OR1) [15]. Polymerase mutator alleles have been described previously [16] [17] [18]. Heterozygous EXO1/exo1, SWR1/swr1, HTZ1/htz1, and MSH2/msh2 diploids were generated in wild type or polymerase mutator backgrounds by PCR-based targeted gene deletion of the coding region. "
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    ABSTRACT: The yeast SWR-C chromatin remodeling enzyme catalyzes chromatin incorporation of the histone variant H2A.Z which plays roles in transcription, DNA repair, and chromosome segregation. Dynamic incorporation of H2A.Z by SWR-C also enhances the ability of exonuclease I (Exo1) to process DNA ends during repair of double strand breaks. Given that Exo1 also participates in DNA replication and mismatch repair, here we test whether SWR-C influences DNA replication fidelity. We find that inactivation of SWR-C elevates the spontaneous mutation rate of a strain encoding a L612M variant of DNA polymerase (Pol) δ, with a single base mutation signature characteristic of lagging strand replication errors. However, this genomic instability does not solely result from reduced Exo1 function, because single base mutator effects are seen in both Exo1-proficient and Exo1-deficient pol3-L612M swr1Δ strains. The data are consistent with the possibility that incorporation of the H2A.Z variant by SWR-C may stimulate Exo1 activity, as well as enhance the fidelity of replication by Pol δ, the repair of mismatches generated by Pol δ, or both. Copyright © 2014 Elsevier B.V. All rights reserved.
    DNA Repair 11/2014; 25C:9-14. DOI:10.1016/j.dnarep.2014.10.010 · 3.11 Impact Factor
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    • "Here we have developed the eSPAN method and presented the following lines of evidence supporting the idea that the eSPAN method can reveal whether a protein is enriched at leading or lagging strands of DNA replication forks. First, we show that the catalytic subunits of Polε and Pold associate preferentially with leading and lagging strands, respectively, of both HU-stalled forks and normal forks, providing direct evidence supporting the division of labor of these polymerases during DNA replication (Nick McElhinny et al., 2008; Pursell et al., 2007). Second, we show that Cdc45 and Mcm6, two subunits of active replicative helicase CMG complex, bind preferentially to leading strands of HU-stalled replication forks during the early S phase of the cell cycle. "
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    ABSTRACT: In eukaryotic cells, DNA replication proceeds with continuous synthesis of leading-strand DNA and discontinuous synthesis of lagging-strand DNA. Here we describe a method, eSPAN (enrichment and sequencing of protein-associated nascent DNA), which reveals the genome-wide association of proteins with leading and lagging strands of DNA replication forks. Using this approach in budding yeast, we confirm the strand specificities of DNA polymerases delta and epsilon and show that the PCNA clamp is enriched at lagging strands compared with leading-strand replication. Surprisingly, at stalled forks, PCNA is unloaded specifically from lagging strands. PCNA unloading depends on the Elg1-containing alternative RFC complex, ubiquitination of PCNA, and the checkpoint kinases Mec1 and Rad53. Cells deficient in PCNA unloading exhibit increased chromosome breaks. Our studies provide a tool for studying replication-related processes and reveal a mechanism whereby checkpoint kinases regulate strand-specific unloading of PCNA from stalled replication forks to maintain genome stability. Copyright © 2014 Elsevier Inc. All rights reserved.
    Molecular Cell 11/2014; 56(4). DOI:10.1016/j.molcel.2014.11.007 · 14.02 Impact Factor
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    • "The spectrum of nucleotide changes in POLE-exo* tumors is distinct from the mutation spectrum in MSI and MSS tumors, characterized mainly by a prevalence of TCT!TAT and TCG!TTG mutations (see also Supplemental Fig. S1; The Cancer Genome Atlas Network 2012, 2013; Alexandrov et al. 2013; Kane and Shcherbakova 2014). Studies in yeast based on selected, well-characterized origins of replication (ORI) demonstrated that POLE synthesizes the leading strand (Pursell et al. 2007b; Miyabe et al. 2011), whereas DNA polymerase delta (POLD1), which is encoded by POLD1, synthesizes the lagging strand (Nick McElhinny et al. 2008). "
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    ABSTRACT: Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas group B mutants are found in POLE and POLD1, and appear to be non-functional. In group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication.
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