A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.     

Cyclin F is degraded during G2-M by mechanisms fundamentally different from other cyclins

Cyclin F, a cyclin that can form SCF complexes and bind to cyclin B, oscillates in the cell cycle with a pattern similar to cyclin A and cyclin B. Ectopic expression of cyclin F arrests the cell cycle in G(2)/M. How the level of cyclin F is regulated during the cell cycle is completely obscure. Here we show that, similar to cyclin A, cyclin F is degraded when the spindle assembly checkpoint is activated and accumulates when the DNA damage checkpoint is activated. Cyclin F is a very unstable protein throughout much of the cell cycle. Unlike other cyclins, degradation of cyclin F is independent of ubiquitination and proteasome-mediated pathways. Interestingly, proteolysis of cyclin F is likely to involve metalloproteases. Rapid destruction of cyclin F does not require the N-terminal F-box motif but requires the COOH-terminal PEST sequences. The PEST region alone is sufficient to interfere with the degradation of cyclin F and confer instability when fused to cyclin A. These data show that although cyclin F is degraded at similar time as the mitotic cyclins, the underlying mechanisms are entirely distinct.     

The identification of novel 5′-amino gemcitabine analogs as potent RRM1 inhibitors

The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series. In short, we found that gemcitabine, as its diphosphate metabolite, represents one of the few described active site inhibitors of RRM1. We herein describe the identification of novel 5′-amino gemcitabine analogs as potent RRM1 inhibitors through in-cell phenotypic screening.     

Identification of a family of human F-box proteins.

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.     

SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.     

The F-box protein Dia2 regulates DNA replication

Ubiquitin-mediated proteolysis plays a key role in many pathways inside the cell and is particularly important in regulating cell cycle transitions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases whose specificity is determined by a substrate-binding F-box protein. Dia2 is a Saccharomyces cerevisiae F-box protein previously described to play a role in invasive growth and pheromone response pathways. We find that deletion of DIA2 renders cells cold-sensitive and subject to defects in cell cycle progression, including premature S-phase entry. Consistent with a role in regulating DNA replication, the Dia2 protein binds replication origins. Furthermore, the dia2 mutant accumulates DNA damage in both S and G2/M phases of the cell cycle. These defects are likely a result of the absence of SCF(Dia2) activity, as a Dia2 DeltaF-box mutant shows similar phenotypes. Interestingly, prolonging G1-phase in dia2 cells prevents the accumulation of DNA damage in S-phase. We propose that Dia2 is an origin-binding protein that plays a role in regulating DNA replication.     

αB-crystallin is mutant B-RAF regulated and contributes to cyclin D1 turnover in melanocytic cells

The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, αB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that αB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate αB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. αB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of αB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that αB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, αB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage.     

The novel human DNA helicase hFBH1 is an F-box protein

We have identified a novel DNA helicase in humans that belongs to members of the superfamily I helicase and found that it contains a well conserved F-box motif at its N terminus. We have named the enzyme hFBH1 (human F-box DNA helicase 1). Recombinant hFBH1, containing glutathione S-transferase at the N terminus, was expressed in Sf9 cells and purified. In this report, we show that hFBH1 exhibited DNA-dependent ATPase and DNA unwinding activities that displace duplex DNA in the 3' to 5' direction. The hFBH1 enzyme interacted with human SKP1 and formed an SCF (SKP1/Cullin/F-box) complex together with human Cullin and ROC1. In addition, the SCF complex containing hFBH1 as an F-box protein displayed ubiquitin ligase activity. We demonstrate that hFBH1 is the first F-box protein that possesses intrinsic enzyme activity. The potential role of the F-box motif and the helicase activity of the enzyme are discussed with regard to regulation of DNA metabolism.     

Stable suppression of the R2 subunit of ribonucleotide reductase by R2-targeted short interference RNA sensitizes p53(-/-) HCT-116 colon cancer cells to DNA-damaging agents and ribonucleotide reductase inhibitors

Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(-/-) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. To evaluate the involvement of R2 in response to DNA damage, p53(-/-) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(-/-) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2.     

The cyclin A1-CDK2 complex regulates DNA double-strand break repair

Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53. cyclin A1-/- cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.     

NIPA defines an SCF-type mammalian E3 ligase that regulates mitotic entry

The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-box subunit of the SCF specifically recruiting a given substrate to the SCF core. Here we identify NIPA (nuclear interaction partner of ALK) as a human F-box-containing protein that defines an SCF-type E3 ligase (SCF(NIPA)) controlling mitotic entry. Assembly of this SCF complex is regulated by cell-cycle-dependent phosphorylation of NIPA, which restricts substrate ubiquitination activity to interphase. We show nuclear cyclin B1 to be a substrate of SCF(NIPA). Inactivation of NIPA by RNAi results in nuclear accumulation of cyclin B1 in interphase, activation of cyclin B1-Cdk1 kinase activity, and premature mitotic entry. Thus, SCF(NIPA)-based ubiquitination may regulate S-phase completion and mitotic entry in the mammalian cell cycle.     

Identification of ribonucleotide reductase M2 as a potential target for pro-senescence therapy in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is the leading cause of gynecological-related cancer deaths in the United States. There is, therefore, an urgent need to develop novel therapeutic strategies for this devastating disease. Cellular senescence is a state of stable cell growth arrest that acts as an important tumor suppression mechanism. Ribonucleotide reductase M2 (RRM2) plays a key role in regulating the senescence-associated cell growth arrest by controlling biogenesis of 2'-deoxyribonucleoside 5′-triphosphates (dNTPs). The role of RRM2 in EOC remains poorly understood. Here we show that RRM2 is expressed at higher levels in EOCs compared with either normal ovarian surface epithelium (P < 0.001) or fallopian tube epithelium (P < 0.001). RRM2 expression significantly correlates with the expression of Ki67, a marker of cell proliferation (P < 0.001). Moreover, RRM2 expression positively correlates with tumor grade and stage, and high RRM2 expression independently predicts a shorter overall survival in EOC patients (P < 0.001). To delineate the functional role of RRM2 in EOC, we knocked down RRM2 expression in a panel of EOC cell lines. Knockdown of RRM2 expression inhibits the growth of human EOC cells. Mechanistically, RRM2 knockdown triggers cellular senescence in these cells. Notably, this correlates with the induction of the DNA damage response, a known mediator of cellular senescence. These data suggest that targeting RRM2 in EOCs by suppressing its activity is a novel pro-senescence therapeutic strategy that has the potential to improve survival of EOC patients.     

The human F-Box DNA helicase FBH1 faces Saccharomyces cerevisiae Srs2 and postreplication repair pathway roles

The Saccharomyces cerevisiae Srs2 UvrD DNA helicase controls genome integrity by preventing unscheduled recombination events. While Srs2 orthologues have been identified in prokaryotic and lower eukaryotic organisms, human orthologues of Srs2 have not been described so far. We found that the human F-box DNA helicase hFBH1 suppresses specific recombination defects of S. cerevisiae srs2 mutants, consistent with the finding that the helicase domain of hFBH1 is highly conserved with that of Srs2. Surprisingly, hFBH1 in the absence of SRS2 also suppresses the DNA damage sensitivity caused by inactivation of postreplication repair-dependent functions leading to PCNA ubiquitylation. The F-box domain of hFBH1, which is not present in Srs2, is crucial for hFBH1 functions in substituting for Srs2 and postreplication repair factors. Furthermore, our findings indicate that an intact F-box domain, acting as an SCF ubiquitin ligase, is required for the DNA damage-induced degradation of hFBH1 itself. Overall, our findings suggest that the hFBH1 helicase is a functional human orthologue of budding yeast Srs2 that also possesses self-regulation properties necessary to execute its recombination functions.     

Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.     

Molecular dissection of the APC/C inhibitor Rca1 shows a novel F-box-dependent function

Rca1 (regulator of Cyclin A)/Emi (early mitotic inhibitor) proteins are essential inhibitors of the anaphase-promoting complex/cyclosome (APC/C). In Drosophila, Rca1 is required during G2 to prevent premature cyclin degradation by the Fizzy-related (Fzr)-dependent APC/C activity. Here, we present a structure and function analysis of Rca1 showing that a carboxy-terminal fragment is sufficient for APC/C inhibition. Rca1/Emi proteins contain a conserved F-box and interact with components of the Skp-Cullin-F-box (SCF) complex. So far, no function has been ascribed to this domain. We find that the F-box of Rca1 is dispensable for APC/C-Fzr inhibition during G2. Nevertheless, we show that Rca1 has an additional function at the G1-S transition, which requires the F-box. Overexpression of Rca1 accelerates the G1-S transition in an F-box-dependent manner. Conversely, S-phase entry is delayed in cells in which endogenous Rca1 is replaced by a transgene lacking the F-box. We propose that Rca1 acts as an F-box protein in an as yet uncharacterized SCF complex, which promotes S-phase entry.     

An SCF complex containing Fbxl12 mediates DNA damage-induced Ku80 ubiquitylation

The Ku heterodimer, composed of Ku70 and Ku80, is the initiating factor of the nonhomologous end joining (NHEJ) double-strand break (DSB) repair pathway. Ku is also thought to impede the homologous recombination (HR) repair pathway via inhibition of DNA end resection. Using the cell-free Xenopus laevis egg extract system, we had previously discovered that Ku80 becomes polyubiquitylated upon binding to DSBs, leading to its removal from DNA and subsequent proteasomal degradation. Here we show that the Skp1-Cul1-F box (SCF) E3 ubiquitin ligase complex is required for Ku80 ubiquitylation and removal from DNA. A screen for DSB-binding F box proteins revealed that the F box protein Fbxl12 was recruited to DNA in a DSB- and Ku-sensitive manner. Immunodepletion of Fbxl12 prevented Cul1 and Skp1 binding to DSBs and Ku80 ubiquitylation, indicating that Fbxl12 is the F box protein responsible for Ku80 substrate recognition. Unlike typical F box proteins, the F box of Fbxl12 was essential for binding to both Skp1 and its substrate Ku80. Besides Fbxl12, six other chromatin-binding F box proteins were identified in our screen of a subset of Xenopus F box proteins: β-TrCP, Fbh1, Fbxl19, Fbxo24, Fbxo28 and Kdm2b. Our study unveils a novel function for the SCF ubiquitin ligase in regulating the dynamic interaction between DNA repair machineries and DSBs.     

The contribution of CMP kinase to the efficiency of DNA repair

Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. In this study, we investigated the role of CMP/UMP kinase (CMPK), an enzyme catalyzes CDP formation, in DNA repair. Knockdown of CMPK delays DNA repair during recovery from UV damage in serum-deprived cells but not in the cells without serum deprivation. Exogenous supply of cytidine or deoxycytidine facilitates DNA repair dependent on CMPK in serum-deprived cells, suggesting that the synthesis of dCDP or CDP determines the rate of repair. However, CMPK knockdown does not affect the steady state level of dCTP in serum-deprived cells. We then found the localization of CMPK at DNA damage sites and its complex formation with Tip60 and ribonucleotide reductase. Our analysis demonstrated that the N-terminal 32-amino-acid of CMPK is required for its recruitment to DNA damage sites in a Tip60-dependent manner. Re-expression of wild-type but not N-terminus deleted CMPK restores the efficiency of DNA repair in CMPK knockdown cells. We proposed that site-specific dCDP formation via CMPK provides a means to facilitate DNA repair in serum-deprived cells.     

The contribution of CMP kinase to the efficiency of DNA repair

Cellular supply of deoxynucleoside triphosphates (dNTPs) is crucial for DNA replication and repair. In this study, we investigated the role of CMP/UMP kinase (CMPK), an enzyme catalyzes CDP formation, in DNA repair. Knockdown of CMPK delays DNA repair during recovery from UV damage in serum-deprived cells but not in the cells without serum deprivation. Exogenous supply of cytidine or deoxycytidine facilitates DNA repair dependent on CMPK in serum-deprived cells, suggesting that the synthesis of dCDP or CDP determines the rate of repair. However, CMPK knockdown does not affect the steady state level of dCTP in serum-deprived cells. We then found the localization of CMPK at DNA damage sites and its complex formation with Tip60 and ribonucleotide reductase. Our analysis demonstrated that the N-terminal 32-amino-acid of CMPK is required for its recruitment to DNA damage sites in a Tip60-dependent manner. Re-expression of wild-type but not N-terminus deleted CMPK restores the efficiency of DNA repair in CMPK knockdown cells. We proposed that site-specific dCDP formation via CMPK provides a means to facilitate DNA repair in serum-deprived cells.     

Activation of Deoxyribonucleotide Synthesis by Radioprotectants and Antioxidants as a Key Stage in Formation of Body Resistance to DNA-Damaging Factors

The responses of the systems of synthesis of deoxyribonucleotides (dNTPs), DNA, and proteins in hematopoietic organs and liver of animals to γ-radiation, administration of radioprotectants and antioxidants as well as the dependence of these responses on the doses of radiation and drugs were studied. Radioprotectants of acute (indralin) and durable effects (indomethaphen) as well as natural α₂-tocopherol) and synthetic antioxidants (ionol or 2,6-di-tert-butyl-4-methylphenol) efficient in survival test were used. Three stages could be recognized in the standard unspecific response of the studied systems to radiation: (1) immediate increase in ribonucleotide reductase activity in the tissues within the first 30 min as a part of the integrated SOS response to DNA damage, which activates dNTP synthesis; (2) inhibition of the synthesis of dNTPs, DNA, and proteins; and (3) restoring ribonucleotide reductase activity and integral increase in the production of dNTPs, DNA, and total protein, which is essential for the development of compensatory and restorative responses of the organism. The radioprotectants significantly increased ribonucleotide reductase activity, which increased intracellular concentrations of the four dNTP types in organs during radiation exposure and three following days. Within this period, ribonucleotide reductase activity was inhibited by 40–50% in animals not treated with radioprotectants as compared to control. Balanced high pools of dNTPs in the organs of radioprotectant-treated animals provided for high-performance repair of DNA damage. The radioprotectant-induced activation of dNTP synthesis during the development of compensatory and restorative responses provides for an earlier restoration of the cellular composition and functioning of the organs. Antioxidants stimulated the synthesis of dNTPs, DNA, and proteins in animal tissues in a strict dose interval. Their effect on the studied syntheses was dose-dependent: single or multiple long-term administration of high antioxidant doses inhibited synthesis of dNTPs, DNA, and proteins. Radioprotectants and antioxidants affected the pool of blood protein Fe³⁺-transferrin controlling the synthesis of iron-containing ribonucleotide reductase activity in hematopoietic organs, and hence, the iron-dependent stage in DNA synthesis—dNTP synthesis. Activation of protein synthesis in organs by the studied substances increased the pools of Fe³⁺-transferrin and Cu²⁺-ceruloplasmin in the blood, which activated dNTP and DNA synthesis. Activated synthesis of dNTP, DNA, and proteins in the organs and increased pools of studied plasma proteins underlay the formation of body resistance to DNA-damaging factors.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 401–422.Original Russian Text Copyright © 2005 by Sharygin, Pulatova, Shlyakova, Mitrokhin, Todorov.