The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

Chilkova, Olga; Jonsson, Bengt‐Harald; Johansson, Erik

doi:10.1074/jbc.m211818200

Cited by 67 publications

(76 citation statements)

References 33 publications

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“…Other models for the roles of Pol ␦ and Pol ⑀ in chromosomal replication have also been considered (14,15). "One strand-one polymerase" models are consistent with the observation that both polymerases are monomers in solution (16,17) and with our earlier genetic studies demonstrating that the 3Ј 3 5Ј exonucleases of Pol ␦ and Pol ⑀ correct replication errors on opposite DNA strands (18). However, it is not yet known whether Pol ␦ and Pol ⑀ always correct their own errors in vivo, and genetic data on the roles of Pol ␦ and Pol ⑀ exonucleases are not sufficient to conclude that the two polymerases perform synthesis on opposite DNA strands.…”

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confidence: 74%

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Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

Shcherbakova

Pavlov

Chilkova

et al. 2003

Journal of Biological Chemistry

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113

137

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We have purified wild type and exonuclease-deficient four-subunit DNA polymerase ⑀ (Pol ⑀) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol ⑀ synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of <2 ؋ 10 ؊5 and <5 ؋ 10 ؊7 , respectively. Pol ⑀ lacking 3 3 5 exonuclease activity is less accurate to a degree suggesting that wild type Pol ⑀ proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol ⑀ is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T⅐dTTP, T⅐dCTP, and C⅐dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol ⑀ differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol ⑀ in vitro and substitutions occurring in vivo in a yeast strain defective in Pol ⑀ proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol ⑀ polymerase activity in DNA replication.Replication of chromosomes in eukaryotes is believed to require three DNA polymerases, Pol ␣, 1 Pol ␦, and Pol ⑀ (for reviews, see Refs. 1-3). Pol ␣ has an associated DNA primase activity and synthesizes short RNA-DNA primers to initiate replication at origins and start Okazaki fragments on the lagging DNA strand. The roles of Pol ␦ and Pol ⑀ in chromosomal replication are not clearly defined. Evidence for essential roles of Pol ␦ and Pol ⑀ in replication comes from genetic studies in Saccharomyces cerevisiae. Mutational inactivation of catalytic subunits and some of the additional subunits of both polymerases is lethal. Strains carrying temperature-sensitive mutations in POL3 gene (encoding the catalytic subunit of Pol ␦), POL2, or DPB2 genes (encoding subunits of Pol ⑀) arrest in S phase of the cell cycle with a terminal morphology characteristic of a DNA replication defect upon shift to non-permissive temperature (4 -6). Incorporation of labeled precursors into DNA stops in these mutants at non-permissive temperature (4, 5, 7). The POL3, POL2, and DPB2 genes, as well as the DPB3 gene encoding the third subunit of Pol ⑀, are expressed periodically in the cell cycle with a peak in G 1 to S phase transition (4, 5, 8), which is characteristic of genes encoding DNA replication proteins. Both Pol ␦ and Pol ⑀ have intrinsic 3Ј 3 5Ј exonuclease activities that correct polymerase errors during replication (9, 10). Mutations in POL3 or POL2 inactivating the exonucleases result in a mutator phenotype (11,12).To accommodate roles for Pol ␦ and Pol ⑀ at a replication fork, it was proposed...

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confidence: 74%

“…Wild type Pol ⑀ was overproduced in the POL2 ϩ strain, and the exonuclease-deficient Pol ⑀ with the FDIET 3 FAIAT substitution was overproduced in the pol2-4 strain. The enzymes were purified in parallel by conventional chromatography as described previously (17).…”

Section: Methodsmentioning

confidence: 99%

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

Shcherbakova

Pavlov

Chilkova

et al. 2003

Journal of Biological Chemistry

Self Cite

113

137

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“…These interactions may have diverged in different species, since Schizosaccharomyces pombe DNA pol e catalytic subunit (cdc20) cannot rescue mutations in S. cerevisiae Pol2 and vice versa (Sugino et al, 1998). The native DNA pol e holoenzyme is a heterotetramer (Chilkova et al, 2003). Two of the three smaller subunits can be identified in Arabidopsis with a BLAST search.…”

Section: Online)mentioning

confidence: 99%

Interactions between the Cell Cycle and Embryonic Patterning inArabidopsisUncovered by a Mutation in DNA Polymerase ε

2005

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Pattern formation and morphogenesis require coordination of cell division rates and orientations with developmental signals that specify cell fate. A viable mutation in the TILTED1 locus, which encodes the catalytic subunit of DNA polymerase e of Arabidopsis thaliana, causes a lengthening of the cell cycle by ;35% throughout embryo development and alters cell type patterning of the hypophyseal lineage in the root, leading to a displacement of the root pole from its normal position on top of the suspensor. Treatment of preglobular and early globular stages, but not later stage, embryos with the DNA polymerase inhibitor aphidicolin leads to a similar phenotype. The results uncover an interaction between the cell cycle and the processes that determine cell fate during plant embryogenesis.

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“…Most of the major replicative DNA polymerases, often called replicases, are multi-subunit holoenzymes (HE). In eukaryotic cells, DNA replication is executed by at least three DNA polymerases: Pol a, Pol d, and Pol e. Pol e HE is a four-subunit complex composed of a DNA polymerase/exonuclease subunit, Pol2p, and three auxiliary subunits: Dpb2p, Dpb3p, and Dpb4p Dua et al 2000;Chilkova et al 2003;Asturias et al 2006). This composition of subunits is conserved from yeast to humans (Kesti et al 1993;Li et al 1997;Jokela et al 1998;Li et al 2000;Feng et al 2003;Spiga and D'Urso 2004).…”

mentioning

confidence: 99%

Dpb2p, a Noncatalytic Subunit of DNA Polymerase ε, Contributes to the Fidelity of DNA Replication in Saccharomyces cerevisiae

et al. 2008

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Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperaturesensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 39 / 59 proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 39 / 59 exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.

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The Quaternary Structure of DNA Polymerase ε from Saccharomyces cerevisiae

Cited by 67 publications

References 33 publications

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

Unique Error Signature of the Four-subunit Yeast DNA Polymerase ϵ

Interactions between the Cell Cycle and Embryonic Patterning inArabidopsisUncovered by a Mutation in DNA Polymerase ε

Dpb2p, a Noncatalytic Subunit of DNA Polymerase ε, Contributes to the Fidelity of DNA Replication in Saccharomyces cerevisiae

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