Control of dTTP pool size by anaphase promoting complex/cyclosome is essential for the maintenance of genetic stability

Ke, Po-Yuan; Kuo, Yueh‐Hsiung; Hu, Chuan-Mei; Chang, Zee-Fen

doi:10.1101/gad.1322905

Cited by 78 publications

(71 citation statements)

References 41 publications

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“…Materials and Antibodies-Anti-human TK1 polyclonal antibody (13,40) and the anti-human TK1 monoclonal antibody (XPA 210) (41) were described previously. Anti-human TS antibody (clone 4H4B1) was obtained from Zymed Laboratories Inc., anti-R2 (N18), anti-cyclin B1 (GNS1), anti-chk1 (G-4), anti-p21(187), and anti-p53R2(N16) antibodies were from Santa Cruz, anti-phospho-chk1 (Ser-345) antibody was from Cell Signaling, anti-phospho-histone 3 (H3; Ser-10) and anti-phospho-H2A.X (Ser-139) antibodies were from Upstate, anti-p53 (Ab-6) antibody was from Calbiochem.…”

Section: Methodsmentioning

confidence: 99%

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Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Chen

Eriksson

Chang

2010

Journal of Biological Chemistry

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Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G 2 phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G 1 /S progression, resulting in a smaller G 2 fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53 ؊/؊ cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G 2 arrest.The synthesis of dTTP is highly regulated and is important for DNA replication and repair in all living cells (1, 2). There are two pathways for dTTP synthesis in cells. In the de novo pathway, ribonucleotide reductase, which is composed of two pairs of R1 and R2 subunits, converts CDP and UDP to dCDP and dUDP, respectively. Both dCDP and dUDP can be metabolically converted to dUMP, and thymidylate synthase (TS) 3 catalyzes the reaction of dTMP formation from dUMP. In the salvage pathway, thymidine kinases (TKs), TK1 in cytosol and TK2 in mitochondria (3, 4), are responsible for dTMP production from thymidine (5). Phosphorylation of dTMP from either the salvage or de novo pathway to dTDP is catalyzed by thymidylate kinase, and nucleoside diphosphate kinase then converts dTDP to dTTP (6). Unlike TK2, the expression of TK1, TS, and thymidylate kinase is cell cycle-dependent (7-13).In response to DNA damage, cells trigger multifaceted responses such as cell cycle arrest, DNA repair, or apoptosis (14, 15). In Saccharomyces cerevisiae, it has been reported that DNA damage by ␥-irradiation, UV, or methyl methane sulfonate leads to increases in the levels of the four dNTPs through ribonucleotide reductase-mediated de novo synthesis, indicating a close relationship between the regulation of dNTP synthesis and DNA damage response (16,17). In mammalian cells, expression of p53-inducible R2 (p53R2), a homolog of the R2 subunit, is increased due to p53-dependent transcriptional activation upon DNA damage, suggesting a master role of p53 in integrating regulation of dNTP pools through the de novo pathway (18 -21). However, it is known that more than 50% human cancer cells harbor mutated or deleted p53, and these tumors are more resistant to chemotherapy due to the loss of p53-dependent apoptosis (22,23). This evoked the question as how p53-deficie...

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Section: Methodsmentioning

confidence: 99%

“…Phosphorylation of dTMP from either the salvage or de novo pathway to dTDP is catalyzed by thymidylate kinase, and nucleoside diphosphate kinase then converts dTDP to dTTP (6). Unlike TK2, the expression of TK1, TS, and thymidylate kinase is cell cycle-dependent (7)(8)(9)(10)(11)(12)(13).…”

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Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Chen

Eriksson

Chang

2010

Journal of Biological Chemistry

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“…T he maintenance of adequate and balanced dNTP levels is critical for faithful DNA replication and repair and for the survival of all organisms (1)(2)(3)(4)(5). A major target of dNTP pool regulation is ribonucleotide reductase (RNR) that catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms in dNTP biosynthesis (6).…”

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Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1

Zhang

Yang

Chen

et al. 2007

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

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Ribonucleotide reductase maintains cellular deoxyribonucleotide pools and is thus tightly regulated during the cell cycle to ensure high fidelity in DNA replication. The Sml1 protein inhibits ribonucleotide reductase activity by binding to the R1 subunit. At the completion of each turnover cycle, the active site of R1 becomes oxidized and subsequently regenerated by a cysteine pair (CX 2C) at its C-terminal domain (R1-CTD). Here we show that R1-CTD acts in trans to reduce the active site of its neighboring monomer. Both Sml1 and R1-CTD interact with the N-terminal domain of R1 (R1-NTD), which involves a conserved two-residue sequence motif in the R1-NTD. Mutations at these two positions enhancing the Sml1-R1 interaction cause SML1-dependent lethality. These results point to a model whereby Sml1 competes with R1-CTD for association with R1-NTD to hinder the accessibility of the CX 2C motif to the active site for R1 regeneration.deoxyribonucleotides ͉ DNA replication T he maintenance of adequate and balanced dNTP levels is critical for faithful DNA replication and repair and for the survival of all organisms (1-5). A major target of dNTP pool regulation is ribonucleotide reductase (RNR) that catalyzes the essential step of converting ribonucleoside diphosphates to the corresponding deoxy forms in dNTP biosynthesis (6). Class I RNRs are conserved from eubacteria to eukaryotes and comprise two subunits: R1 and R2. R1 (␣ 2 in Escherichia coli and ␣ 2 , ␣ 4 , and ␣ 6 in eukaryotes; ref. 7) contains the active site as well as binding sites for allosteric effectors; R2 (␤ 2 ) houses a diferric-tyrosyl radical required for catalysis (8). These subunits can be regulated by allostery (1), transcription (9), subcellular compartmentalization (10 -13), and protein inhibitor interaction (14, 15).The 104-residue Saccharomyces cerevisiae Sml1 protein was originally identified as an RNR inhibitor based on the finding that loss of SML1 function suppresses the lethality of cells lacking the checkpoint kinases Mec1 or Rad53 by increasing cellular dNTP levels (15). Sml1 is phosphorylated and degraded during S phase and after DNA damage in a checkpointdependent manner to relieve RNR inhibition (16). The inhibition of R1 by Sml1 depends on Sml1-R1 association because mutations in SML1 disrupting its R1-binding ability abolish the inhibition (17). Crystallographic studies of the R1s from E. coli and S. cerevisiae reveal three domains in the protein: the N-terminal helical domain, the 10-stranded ␣/␤-barrel domain, and the C-terminal domain of less-defined structure (18,19). The active site is located in the center of the protein between the N-terminal and the barrel domains, wherein a redox-active cysteine pair (Cys-225/Cys-462 of the E. coli R1 and Cys-218/ Cys-443 of the yeast R1) converts from a free dithiol form in the reduced R1 (active form) to a disulfide-bonded form in the oxidized R1 (inactive form) after each reduction cycle (20). This disulfide bond is reduced to regenerate an active R1 for the subsequent catalytic cycles (21...

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“…Anti-hTMPK and anti-hTK1 polyclonal antibodies were prepared as described previously (23,24). Anti-hTS antibody (clone 4H4B1) and anti-p53 monoclonal antibody (ab-6) were purchased from Zymed and Calbiochem, respectively.…”

Section: Methodsmentioning

confidence: 99%

Synthetic Lethality by Lentiviral Short Hairpin RNA Silencing of Thymidylate Kinase and Doxorubicin in Colon Cancer Cells Regardless of the p53 Status

Chang

2008

Cancer Research

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Intracellular supply of dTTP is a highly regulated process and has been a key target for chemotherapeutic drug development. Thymidylate kinase (TMPK) is the key enzyme for dTTP formation in both de novo and salvage pathways. In this study, we used lentiviral-based small hairpin RNA to silence TMPK expression in p53(+/+) and p53(À/À) HCT-116 colon cancer cells. This approach was sufficient to decrease the dTTP pool gradually without affecting p53 expression and generating cytotoxicity. TMPK knockdown significantly increased doxorubicin sensitivity dramatically in p53-proficient, p53-null HCT-116, and LoVo colon cancer cells. The decrease in the dTTP pool using this approach augmented the DNA damage response and enhanced apoptotic induction after exposure to low-dose doxorubicin, leading to cell death. In contrast, silencing of thymidylate synthase which blocks the de novo pathway was incapable of sensitizing p53-null HCT-116 cells to doxorubicin-induced apoptosis because of the compensation by the salvage pathway. Our results suggest the lentiviral delivery of small hairpin RNA targeting TMPK in combination with a low dose of doxorubicin as a new approach to kill colon cancer cells regardless of p53 status. [Cancer Res 2008;68(8):2831-40]

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Control of dTTP pool size by anaphase promoting complex/cyclosome is essential for the maintenance of genetic stability

Cited by 78 publications

References 41 publications

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1

Synthetic Lethality by Lentiviral Short Hairpin RNA Silencing of Thymidylate Kinase and Doxorubicin in Colon Cancer Cells Regardless of the p53 Status

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