Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

Morrison, Alan; Johnson, Andrew L.; Johnston, Leland H.; Sugino, Akio

doi:10.1002/j.1460-2075.1993.tb05790.x

Cited by 246 publications

(309 citation statements)

References 34 publications

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“…The inviability of pol3-R696W could be caused by (i) severely impaired catalytic activity of Polδ or (ii) the accumulation of lethal mutations in other essential genes because of a dramatic decrease in the fidelity of Polδ. Such a "mutation catastrophe" has been previously described for yeast that are defective simultaneously in two replication error-avoidance mechanisms (exonucleolytic DNA polymerase proofreading and MMR) (17) and for E. coli carrying a defective exonuclease subunit of Pol III (18). If pol3-R696W encodes a nonfunctional polymerase, haploid cells expressing the mutant allele would not be able to replicate their DNA and divide, as observed previously (19) and in this study (Fig.…”

Section: Resultssupporting

confidence: 81%

“…In the mutation-catastrophe scenario, lethal mutations would accumulate during replication, and pol3-R696W cells would be expected to proceed through a number of cell cycles before cell division ceases. This was previously observed for the proofreadingand MMR-deficient yeast cells (17). Microscopic examination after tetrad dissection showed that the pol3-R696W cells were able to divide and form microcolonies of varying size (estimated at ∼100 cells on average) before cell division stopped (Fig.…”

Section: Resultssupporting

confidence: 78%

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A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

Daee

Mertz

Shcherbakova

2009

Proc. Natl. Acad. Sci. U.S.A.

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Accurate DNA synthesis by the replicative DNA polymerases α, δ, and ε is critical for genome stability in eukaryotes. In humans, over 20 SNPs were reported that result in amino-acid changes in Polδ or Polε. In addition, Polδ variants were found in colon-cancer cell lines and in sporadic colorectal carcinomas. Using the yeast-model system, we examined the functional consequences of two cancerassociated Polδ mutations and four polymorphisms affecting wellconserved regions of Polδ or Polε. We show that the R696W substitution in Polδ (analog of the R689W change in the human cancer-cell line DLD-1) is lethal in haploid and homozygous diploid yeast. The cell death results from a catastrophic increase in spontaneous mutagenesis attributed to low-fidelity DNA synthesis by Polδ-R696W. Heterozygotes survive, and the mutation rate depends on the relative expression level of wild-type versus mutant alleles. Based on these observations, we propose that the mutation rate in heterozygous human cells could be regulated by transient changes in gene expression leading to a temporary excess of Polδ-R689W. The similarities between the mutational spectra of the yeast strains producing Polδ-R696W and DLD-1 cells suggest that the altered Polδ could be responsible for a significant proportion of spontaneous mutations in this cancer cell line. These results suggest that the highly error-prone Polδ-R689W could contribute to cancer initiation and/or progression in humans.

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Section: Resultssupporting

confidence: 81%

Section: Resultssupporting

confidence: 78%

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

Daee

Mertz

Shcherbakova

2009

Proc. Natl. Acad. Sci. U.S.A.

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“…In yeast 22,23 and mice 7,16 , expression of pol-d mutants analogous to those studied here brought about an increase in spontaneous mutation rates and, in the mouse, predisposition to cancer. These results suggested that the MMR system was unable to deal with the increased load of mutations, similarly to what was observed in E. coli 20 .…”

Section: Resultsmentioning

confidence: 65%

Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system

Ghodgaonkar

Ventura

et al. 2014

Nat Commun

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Next-generation sequencing has revolutionized the search for disease-causing genetic alterations. Unfortunately, the task of distinguishing the handful of causative mutations from rare variants remains daunting. We now describe an assay that permits the analysis of all types of mutations in any gene of choice through the generation of stable human cell lines, in which the endogenous protein has been inducibly replaced with its genetic variant. Here we studied the phenotype of variants of the essential replicative polymerase-d carrying missense mutations in its active site, similar to those recently identified in familial colon cancer patients. We show that expression of the mutants but not the wild-type protein endows the engineered cells with a mutator phenotype and that the mutations affect the fidelity and/or the exonuclease activity of the isolated enzyme in vitro. This proof-of-principle study demonstrates the general applicability of this experimental approach in the study of genotype-phenotype correlations.

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“…Indeed, in the yeast mutants in which pol delta or epsilon proofreading is selectively inactivated, the mutations rates are 10-100 times higher than the wild-type strains. [26][27][28] Recently, mice carrying artificial alleles with substitutions at essential amino-acid residues in the proofreading domains of pol delta 29,30 and epsilon 31 have been reported. The observed mutation rates were indeed significantly elevated over the wild-type levels.…”

Section: Introductionmentioning

confidence: 99%

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

et al. 2010

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Genomic sequences encoding the 3¢ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms. Keywords: polymerase delta; polymerase epsilon; proofreading domain; colorectal cancer INTRODUCTION Mutation rates on the genome are invariably regulated and typically suppressed to an extremely low level, such as 10 À10 per base replicated, in the organisms. 1 The high fidelity of DNA replication is achieved by several molecular mechanisms. The frequency of erroneous nucleotide incorporation is indeed extremely low compared with that expected from base-pairing energetics, and the vast reduction of the misincorporation frequency involves three steps of molecular events: (a) correct incorporation of complementary nucleotides by DNA polymerases, (b) removal of the newly added nucleotides, particularly incorrectly paired nucleotides, by the 3¢ exonuclease activities associated with the DNA polymerases (proofreading) and (c) postreplicative scanning of mispaired bases by DNA mismatch repair (MMR). 2,3 Various Escherichia coli (E. coli) mutator strains have been used to study these molecular mechanisms by which organisms maintain their mutation rates at very low levels. The dnaQ + (mutD + ) gene of E. coli encodes the epsilon subunit of the DNA polymerase III holoenzyme that is involved in 3¢ exonuclease proofreading activity, 4 and, therefore, the mutation frequencies in the dnaQ mutators are sometimes 1000-10 000 times higher than the wild-type levels. 4,5 In E. coli, MMR is chiefly directed by the proteins encoded by the three genes: mutS + , mutL + and mutH + . 6 The mutS or mutL mutators exhibit an approximately 100 times increase in the mutation frequency. 7 Thus, polymerase proofreading and MMR are the two major

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Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

Cited by 246 publications

References 34 publications

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

A cancer-associated DNA polymerase δ variant modeled in yeast causes a catastrophic increase in genomic instability

Phenotypic characterization of missense polymerase-δ mutations using an inducible protein-replacement system

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

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