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: 14.46). 05/2008; 30(2):137-44. DOI: 10.1016/j.molcel.2008.02.022
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Carrie M Stith, Mar 13, 2015
1 Follower
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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 · 3.36 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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 · 14.46 Impact Factor
  • Source
    • "Frontiers in Microbiology | Evolutionary and Genomic Microbiology June 2014 | Volume 5 | Article 305 | 2 Burgers, 2008; Nick McElhinny et al., 2008; Miyabe et al., 2011 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Next-generation sequencing (NGS) technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. Escherichia coli DNA polymerase I proteolytic (Klenow) fragment was originally utilized in Sanger's dideoxy chain-terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE) and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.
    Frontiers in Microbiology 06/2014; 5:305. DOI:10.3389/fmicb.2014.00305 · 3.94 Impact Factor
Show more