Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro

Martomo, Stella A.; Mathews, Christopher K.

doi:10.1016/s0027-5107(01)00283-4

Cited by 48 publications

(36 citation statements)

References 27 publications

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“…Presumably, this fluctuation is important for maintaining genome stability, since reports have shown that elevation of dTTP concentration increases in vitro and in vivo mutation rates (10,29). Extending from these observations, it is noteworthy that dTTP has been identified as an allosteric effector for ribonucleotide reductase, directing its substrate specificity (7).…”

Section: Discussionmentioning

confidence: 99%

Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome–Cdh1-Mediated Pathway

Chang

2004

Molecular and Cellular Biology

109

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The expression of human thymidine kinase 1 (hTK1) is highly dependent on the growth states and cell cycle stages in mammalian cells. The amount of hTK1 is significantly increased in the cells during progression to the S and M phases, and becomes barely detectable in the early G 1 phase by a proteolytic control during mitotic exit. This tight regulation is important for providing the correct pool of dTTP for DNA synthesis at the right time in the cell cycle. Here, we investigated the mechanism responsible for mitotic degradation of hTK1. We show that hTK1 is degraded via a ubiquitin-proteasome pathway in mammalian cells and that anaphasepromoting complex/cyclosome (APC/C) activator Cdh1 is not only a necessary but also a rate-limiting factor for mitotic degradation of hTK1. Furthermore, a KEN box sequence located in the C-terminal region of hTK1 is required for its mitotic degradation and interaction capability with Cdh1. By in vitro ubiquitinylation assays, we demonstrated that hTK1 is targeted for degradation by the APC/C-Cdh1 ubiquitin ligase dependent on this KEN box motif. Taken together, we concluded that activation of the APC/C-Cdh1 complex during mitotic exit controls timing of hTK1 destruction, thus effectively minimizing dTTP formation from the salvage pathway in the early G 1 phase of the cell cycle in mammalian cells.

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Section: Discussionmentioning

confidence: 99%

Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome–Cdh1-Mediated Pathway

Chang

2004

Molecular and Cellular Biology

109

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“…However, the actual pool sizes determined, in pmol/cell, were comparable to values previously reported for cultured mammalian cells. 33,38,39 FACS analysis. Cells were pelleted, and the pellet was resuspended in 500 μl of phosphate-buffered saline containing 0.5% fetal calf serum and fixed in 70% ethanol at -20°C overnight.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, it has been shown by one of our groups that mutational rates increased several fold when the amounts of dNTPs added to the in vitro replication system with HeLa cytoplasmic extracts, were symmetrically increased 3 to 4-fold. 33 Until now, C-MYC has been implicated in the induction of genomic instability largely via its ability to induce intracellular reactive oxygen species. 34 Since it is unclear whether the induction of ROS completely accounts for C-MYC induced mutagenesis, 35 our data raise an intriguing possibility that C-MYC could promote mutagenesis through a nearly symmetrical increase in the amounts of intracellular dNTPs.…”

Section: © 2 0 0 8 L a N D E S B I O S C I E N C E D O N O T D I S mentioning

confidence: 99%

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

Mannava¹,

Grachtchouk²,

Wheeler³

et al. 2008

Cell Cycle

207

189

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To identify C-MYC targets rate-limiting for proliferation of malignant melanoma, we stably inhibited C-MYC in several human metastatic melanoma lines via lentivirus-based shRNAs approximately to the levels detected in normal melanocytes. C-MYC depletion did not significantly affect levels of E2F1 protein reported to regulate expression of many S-phase specific genes, but resulted in the repression of several genes encoding enzymes rate-limiting for dNTP metabolism. These included thymidylate synthase (TS), inosine monophosphate dehydrogenase 2 (IMPDH2) and phosphoribosyl pyrophosphate synthetase 2 (PRPS2). C-MYC depletion also resulted in reduction in the amounts of deoxyribonucleoside triphosphates (dNTPs) and inhibition of proliferation. shRNA-mediated suppression of TS, IMPDH2 or PRPS2 resulted in the decrease of dNTP pools and retardation of the cell cycle progression of melanoma cells in a manner similar to that of C-MYC-depletion in those cells. Reciprocally, concurrent overexpression of cDNAs for TS, IMPDH2 and PRPS2 delayed proliferative arrest caused by inhibition of C-MYC in melanoma cells. Overexpression of C-MYC in normal melanocytes enhanced expression of the above enzymes and increased individual dNTP pools. Analysis of in vivo C-MYC interactions with TS, IMPDH2 and PRPS2 genes confirmed that they are direct C-MYC targets. Moreover, all three proteins express at higher levels in cells from several metastatic melanoma lines compared to normal melanocytes. Our data establish a novel functional link between C-MYC and dNTP metabolism and identify its role in proliferation of tumor cells.

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“…Imbalances in dNTP pools can result in errors in DNA replication, such as single-base substitutions, insertions, or deletions, resulting in frameshift mutagenesis (20). The mismatch repair (MMR) system plays a role in correcting DNA mismatches during replication (21) and thus may contribute to dFdCyd radiosensitization.…”

Section: Introductionmentioning

confidence: 99%

Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers

Flanagan

Robinson

Krokosky

et al. 2007

Molecular Cancer Therapeutics

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Radiation sensitization by 2 ¶,2 ¶-difluoro-2 ¶-deoxycytidine (dFdCyd) has correlated with dATP depletion [dFdCDPmediated inhibition of ribonucleotide reductase (RR)] and S-phase accumulation. We hypothesized that radiosensitization by dFdCyd is due to nucleotide misincorporations in the presence of deoxynucleotide triphosphate pool imbalances, which, if not repaired, augments cell death following irradiation. The ability of dFdCyd to produce misincorporations was measured as pSP189 plasmid mutations in hMLH1-deficient [mismatch repair (MMR) deficient] and hMLH1-expressing (MMR proficient) HCT116 cells. Only MMR-deficient cells showed a significant increase in nucleotide misincorporations (2-to 3-fold increase; P V 0.01) after radiosensitizing concentrations of dFdCyd F 5 Gy radiation, which persisted for at least 96 h. dFdCyd (10 nmol/L) did not radiosensitize MMR-proficient HCT116 or A549 cells, but following small interfering RNA -mediated suppression of hMLH1, this concentration produced excellent radiosensitization (radiation enhancement ratios = 1.6 F 0.1 and 1.5 F 0.1, respectively; P < 0.05) and a 2.5-fold increase in mutation frequency in A549 cells. Cytosine arabinoside (1-B-D-arabinofuranosylcytosine), which can be incorporated into DNA but does not inhibit RR, failed to radiosensitize MMR-deficient cells or increase mutation frequency in the MMR-deficient and MMR-proficient cells. However, the RR inhibitor hydroxyurea radiosensitized MMR-deficient cells and increased nucleotide misincorporations (z5-fold increase; P < 0.05), thus further implicating the inhibition of RR as the mechanism underlying radiosensitization by dFdCyd. These data showed that the presence and persistence of mismatched nucleotides is integral to radiosensitization by dFdCyd and suggest a role for hMLH1 deficiency in eliciting the radiosensitizing effect.

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Effects of biological DNA precursor pool asymmetry upon accuracy of DNA replication in vitro

Cited by 48 publications

References 27 publications

Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome–Cdh1-Mediated Pathway

Mitotic Degradation of Human Thymidine Kinase 1 Is Dependent on the Anaphase-Promoting Complex/Cyclosome–Cdh1-Mediated Pathway

Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells

Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers

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