Faculty Opinions recommendation of Yeast DNA polymerase epsilon participates in leading-strand DNA replication.

The CMG complex (Cdc45, Mcm2–7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

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Detection and characterization of replication origins defined by DNA polymerase epsilon

10.1101/2021.07.27.453931 ◽

2021 ◽

Author(s):

Roman Jaksik ◽

David A Wheeler ◽

Marek Kimmel

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

The Cancer Genome Atlas ◽

Detection Methods ◽

Whole Genome Sequencing Data ◽

Replication Origins ◽

Sequencing Data ◽

Dna Polymerase Epsilon ◽

Polymerase Epsilon ◽

Viable Approach

Human origins of replication (ORI) are recognized by the cellular machinery through mechanisms which are still poorly understood. The process of DNA replication is highly conserved, but the location of ORI may vary considerably from one round of replication to the next, and depends on many factors, making them difficult to map in the genome. We investigated the possibility that at genomic scale, the mutator phenotype associated with DNA polymerase epsilon; exonuclease domain mutation could identify genomic positions of replication origins. Here we report the genome-wide localization of DNA replication origins in POLE-mutated tumors using whole genome sequencing data from The Cancer Genome Atlas project. We show that mutation-based detection of replication origins allows to identify constitutive origins shared between various individuals. We developed a novel method to compare two or more sets of genomic coordinates based on Smith-Waterman-like dynamic programming, which we used to compare replication origin positions obtained using multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods. Many DNA features co-localized with ORI, including chromatin loop anchors, G-quadruplexes, S/MARs and CpGs. Among all features, the H2A.Z histone exhibited the most significant association. Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features.

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Mutations in the Non-Catalytic Subunit Dpb2 of DNA Polymerase Epsilon Affect the Nrm1 Branch of the DNA Replication Checkpoint

PLoS Genetics ◽

10.1371/journal.pgen.1006572 ◽

2017 ◽

Vol 13 (1) ◽

pp. e1006572 ◽

Cited By ~ 6

Author(s):

Michał Dmowski ◽

Justyna Rudzka ◽

Judith L. Campbell ◽

Piotr Jonczyk ◽

Iwona J. Fijałkowska

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Catalytic Subunit ◽

Replication Checkpoint ◽

Dna Polymerase Epsilon ◽

Dna Replication Checkpoint ◽

Polymerase Epsilon

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DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.496 ◽

1993 ◽

Vol 13 (1) ◽

pp. 496-505 ◽

Cited By ~ 64

Author(s):

M E Budd ◽

J L Campbell

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Dna Polymerases ◽

Nonpermissive Temperature ◽

Dna Polymerase Delta ◽

Dna Polymerase Epsilon ◽

Dna Polymerase Alpha ◽

Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

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DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.496-505.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 496-505

Author(s):

M E Budd ◽

J L Campbell

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Dna Polymerases ◽

Nonpermissive Temperature ◽

Dna Polymerase Delta ◽

Dna Polymerase Epsilon ◽

Dna Polymerase Alpha ◽

Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

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DNA polymerization-independent functions of DNA polymerase epsilon in assembly and progression of the replisome in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e12-05-0339 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3240-3253 ◽

Cited By ~ 28

Author(s):

Tetsuya Handa ◽

Mai Kanke ◽

Tatsuro S. Takahashi ◽

Takuro Nakagawa ◽

Hisao Masukata

Keyword(s):

Fission Yeast ◽

Dna Polymerase ◽

Temperature Sensitive ◽

Replication Forks ◽

Dna Polymerization ◽

Dna Polymerase Epsilon ◽

Polymerase Domain ◽

Independent Functions ◽

Leading Strand ◽

Polymerase Epsilon

DNA polymerase epsilon (Pol ε) synthesizes the leading strands, following the CMG (Cdc45, Mcm2-7, and GINS [Go-Ichi-Nii-San]) helicase that translocates on the leading-strand template at eukaryotic replication forks. Although Pol ε is essential for the viability of fission and budding yeasts, the N-terminal polymerase domain of the catalytic subunit, Cdc20/Pol2, is dispensable for viability, leaving the following question: what is the essential role(s) of Pol ε? In this study, we investigated the essential roles of Pol ε using a temperature-sensitive mutant and a recently developed protein-depletion (off-aid) system in fission yeast. In cdc20-ct1 cells carrying mutations in the C-terminal domain of Cdc20, the CMG components, RPA, Pol α, and Pol δ were loaded onto replication origins, but Cdc45 did not translocate from the origins, suggesting that Pol ε is required for CMG helicase progression. In contrast, depletion of Cdc20 abolished the loading of GINS and Cdc45 onto origins, indicating that Pol ε is essential for assembly of the CMG complex. These results demonstrate that Pol ε plays essential roles in both the assembly and progression of CMG helicase.

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