Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase

Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University Medical School, Nagoya 467-8601, Japan.
Genes & development (Impact Factor: 10.8). 02/2010; 24(4):333-8. DOI: 10.1101/gad.1863810
Source: PubMed


A balanced deoxyribonucleotide (dNTP) supply is essential for DNA repair. Here, we found that ribonucleotide reductase (RNR) subunits RRM1 and RRM2 accumulated very rapidly at damage sites. RRM1 bound physically to Tip60. Chromatin immunoprecipitation analyses of cells with an I-SceI cassette revealed that RRM1 bound to a damage site in a Tip60-dependent manner. Active RRM1 mutants lacking Tip60 binding failed to rescue an impaired DNA repair in RRM1-depleted G1-phase cells. Inhibition of RNR recruitment by an RRM1 C-terminal fragment sensitized cells to DNA damage. We propose that Tip60-dependent recruitment of RNR plays an essential role in dNTP supply for DNA repair.

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    • "NHEJ increases the production of gross CA , such as translocations and radial exchange figures , but it allows cell survival and progression toward mitosis while limit - ing DSBs in the DNA [ Niida , 2010a , b ] . The inhibition of RNR by HU in the sequential treatment with MMC - HU may interfere with this NHEJ repair processing and result in the observed overall increase in the frequency of non - rejoined DSB and the decline in the frequency of rejoined CA ( radial figures ) ( Table III ) . "
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    ABSTRACT: Fanconi's anemia (FA) is a recessive disease; 16 genes are currently recognized in FA. FA proteins participate in the FA/BRCA pathway that plays a crucial role in the repair of DNA damage induced by crosslinking compounds. Hydroxyurea (HU) is an agent that induces replicative stress by inhibiting ribonucleotide reductase (RNR), which synthesizes deoxyribonucleotide triphosphates (dNTPs) necessary for DNA replication and repair. HU is known to activate the FA pathway; however, its clastogenic effects are not well characterized. We have investigated the effects of HU treatment alone or in sequential combination with mitomycin-C (MMC) on FA patient-derived lymphoblastoid cell lines from groups FA-A, B, C, D1/BRCA2, and E and on lymphocytes from two unclassified FA patients. All FA cells showed a significant increase (P < 0.05) in chromosomal aberrations following treatment with HU during the last 3 h before mitosis. Furthermore, when FA cells previously exposed to MMC were treated with HU, we observed an increase of MMC-induced DNA damage that was characterized by high occurrence of DNA breaks and a reduction in rejoined chromosomal aberrations. These findings show that exposure to HU during G2 induces chromosomal aberrations by a mechanism that is independent of its well-known role in replication fork stalling during S-phase and that HU interfered mainly with the rejoining process of DNA damage. We suggest that impaired oxidative stress response, lack of an adequate amount of dNTPs for DNA repair due to RNR inhibition, and interference with cell cycle control checkpoints underlie the clastogenic activity of HU in FA cells. Environ. Mol. Mutagen., 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 02/2015; 56(5). DOI:10.1002/em.21938 · 2.63 Impact Factor
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    • "The production of dUTP requires ribonucleotide reductase (RNR), a heteromultimer composed of large (R1) and small (R2) subunits that reduces NDPs to form dNDPs. The subcellular localization of mammalian RNR proteins has been a matter of debate (Pontarin et al., 2008), although recent evidence suggests that at least some RNR complexes localize to sites of DNA damage in the nucleus, where they can contribute to nucleotide production for DNA repair (Niida et al., 2010). Interestingly, the faulty DSB repair following TMPK knockdown could be rescued by disrupting RNR recruitment to DNA lesions, and additional experimental manipulation of RNR expression established RNR levels as a critical determinant of DSB repair proficiency following TMPK impairment. "
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    ABSTRACT: The synthesis of dTDP is unique because there is a requirement for thymidylate kinase (TMPK). All other dNDPs including dUDP are directly produced by ribonucleotide reductase (RNR). We report the binding of TMPK and RNR at sites of DNA damage. In tumor cells, when TMPK function is blocked, dUTP is incorporated during DNA double-strand break (DSB) repair. Disrupting RNR recruitment to damage sites or reducing the expression of the R2 subunit of RNR prevents the impairment of DNA repair by TMPK intervention, indicating that RNR contributes to dUTP incorporation during DSB repair. We identified a cell-permeable nontoxic inhibitor of TMPK that sensitizes tumor cells to doxorubicin in vitro and in vivo, suggesting its potential as a therapeutic option.
    Cancer cell 07/2012; 22(1):36-50. DOI:10.1016/j.ccr.2012.04.038 · 23.52 Impact Factor
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    • "After genotoxic stress, the levels of cyclin F in both p53- positive and p53-negative cells rapidly drop, allowing the recruitment of RRM2 to chromatin for efficient DNA repair synthesis (Figures 6, 7E, S5, and S6). This function is consistent with reports indicating a role for RNR in guaranteeing availability of dNTPs at the sites of DNA damage (Lin et al., 2007; Niida et al., 2010; Zhang et al., 2009). The timing of RRM2 accumulation following DNA damage parallels the timing of DNA repair (Figures 6, 7A, and 7B). "
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    ABSTRACT: F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as an interactor of the F-box protein cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCF(cyclin F) to maintain balanced dNTP pools and genome stability. After DNA damage, cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a nondegradable RRM2 mutant. In summary, we have identified a biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.
    Cell 05/2012; 149(5):1023-34. DOI:10.1016/j.cell.2012.03.043 · 32.24 Impact Factor
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