High incidence of epithelial cancers in mice deficient for DNA polymerase δ proofreading

Goldsby, Robert E.; Hays, Laura E.; Chen, Xin; Olmsted, Elise A.; Slayton, William B.; Spangrude, Gerry J.; Preston, Bradley D.

doi:10.1073/pnas.232340999

Cited by 155 publications

(153 citation statements)

References 43 publications

(49 reference statements)

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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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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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“…1,2 The importance of this 3 0 -5 0 exonuclease activity has been highlighted by genetic analyses in mouse, showing that a point mutation specifically disrupting the 3 0 -5 0 exonuclease proofreading activity of Pold1 causes cancer susceptibility. 6,7 The phenotype of mouse mutants lacking entire Pold1 function, however, has not been reported.…”

Section: Introductionmentioning

confidence: 99%

p53 deficiency rescues apoptosis and differentiation of multiple cell types in zebrafish flathead mutants deficient for zygotic DNA polymerase δ1

et al. 2005

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Cell culture work has identified the tumor suppressor p53 as a component of the S-phase checkpoint control system, while in vivo studies of this role of p53 in whole-vertebrate systems were limited. Here, we describe zebrafish mutants in the DNA polymerase delta catalytic subunit 1, based on the positional cloning of the flathead (fla) gene. fla mutants display specific defects in late proliferative zones, such as eyes, brain and cartilaginous elements of the visceral head skeleton, where cells display compromised DNA replication, followed by apoptosis, and partial or complete loss of affected tissues. Antisense-mediated knockdown of p53 in fla mutants leads to a striking rescue of all phenotypic traits, including completion of replication, survival of cells, and normal differentiation and tissue formation. This indicates that under replicationcompromised conditions, the p53 branch of the S-phase checkpoint is responsible for eliminating stalled cells that, given more time, would have otherwise finished their normal developmental program.

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“…Mutations affecting the fidelity of yeast Polα, Polδ, and Polε result in a spontaneous mutator phenotype (2)(3)(4)(5)(6). In the case of Polδ, when specific nucleotide selectivity and proofreading defects were engineered in mice, the resulting increased mutation rate was also accompanied by an accelerated tumorigenesis (7)(8)(9). This provided support for the mutator hypothesis for cancer, which states that the expression of a mutator phenotype is an early event in tumorigenesis and that it is required for the accumulation of multiple mutations typically observed in cancers (10).…”

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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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High incidence of epithelial cancers in mice deficient for DNA polymerase δ proofreading

Cited by 155 publications

References 43 publications

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

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

p53 deficiency rescues apoptosis and differentiation of multiple cell types in zebrafish flathead mutants deficient for zygotic DNA polymerase δ1

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

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