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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    • "To validate our ability to measure replicative asymmetry using these left-and right-replicating definitions, we considered the one known case of replicative mutational asymmetry: tumors carrying functional mutations in the proofreading exonuclease domain of POLE, the gene encoding polymerase ε (designated as ''POLE tumors'';Shinbrot et al., 2014). The exonuclease domain of polymerase ε is responsible for proofreading during synthesis of the leading strand (Nick McElhinny et al., 2008;Shinbrot et al., 2014), and POLE tumors were previously reported to have high rates of C:G mutations (to A:T or T:A) asymmetrically introduced at cytosines replicated on the leading-strand template near three well-characterized origins of replication (Shinbrot et al., 2014). As a consequence, in these tumors we would expect to see predominantly C/A mutations in left-replicating regions and G/T in right-replicating regions, since we hypothesized these regions to be enriched for leading-and laggingstrand synthesis of the reference strand, respectively. "
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    ABSTRACT: Mutational processes constantly shape the somatic genome, leading to immunity, aging, cancer, and other diseases. When cancer is the outcome, we are afforded a glimpse into these processes by the clonal expansion of the malignant cell. Here, we characterize a less explored layer of the mutational landscape of cancer: mutational asymmetries between the two DNA strands. Analyzing whole-genome sequences of 590 tumors from 14 different cancer types, we reveal widespread asymmetries across mutagenic processes, with transcriptional (“T-class”) asymmetry dominating UV-, smoking-, and liver-cancer-associated mutations and replicative (“R-class”) asymmetry dominating POLE-, APOBEC-, and MSI-associated mutations. We report a striking phenomenon of transcription-coupled damage (TCD) on the non-transcribed DNA strand and provide evidence that APOBEC mutagenesis occurs on the lagging-strand template during DNA replication. As more genomes are sequenced, studying and classifying their asymmetries will illuminate the underlying biological mechanisms of DNA damage and repair.
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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.
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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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