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Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response.

ABSTRACT: To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we conducted synthetic small interfering RNA (siRNA)-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. Silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase complex, respectively, markedly enhanced the cytotoxicity of the topoisomerase I inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by histone gamma-H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels and induces the nuclear translocation of RRM2. The checkpoint kinase 1 (Chk1) was up-regulated and activated in response to CPT, and CHEK1 down-regulation by siRNA and small molecule inhibitors of Chk1 blocked RRM2 induction by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs and DNA repair. Specifically, we propose that upon DNA damage, Chk1 activation, mediated by ATM and ATR, up-regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 expression levels coupled with its nuclear recruitment suggests an active role for ribonucleotide reductase in the cellular response to CPT-mediated DNA damage that could potentially be exploited as a strategy for enhancing the efficacy of topoisomerase I inhibitors.

SUBMITTER: Zhang YW

PROVIDER: S-EPMC2709352 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response.

Zhang Yong-Wei YW Jones Tamara L TL Martin Scott E SE Caplen Natasha J NJ Pommier Yves Y

The Journal of biological chemistry 20090505 27

To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we conducted synthetic small interfering RNA (siRNA)-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. Silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase complex, respectively, markedly enhanced the cytotoxicity of the topoisomerase I inhibitor camptothecin (CPT). Silencing of RRM ...[more]

PMID: 19416980

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Project description:A coenzyme B12-dependent ribonucleotide reductase was purified from the archaebacterium Thermoplasma acidophila and partially sequenced. Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in Escherichia coli. The deduced amino acid sequence shows that the catalytic domain of the B12-dependent enzyme from T. acidophila, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from E. coli, yeast, mammalian viruses, and man. The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not. Evolutionary bridges created by the T. acidophila sequence and that of a B12-dependent reductase from Mycobacterium tuberculosis establish homology between the Fe-dependent enzymes and the catalytic domain of the Lactobacillus leichmannii B12-dependent enzyme as well. These bridges are confirmed by a predicted secondary structure for the Lactobacillus enzyme. Sequence similarities show that the N-terminal domain of the T. acidophila ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from E. coli, which uses neither B12 nor Fe cofactors. A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic E. coli enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B12 ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases. A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment. This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in "post-genomic" biochemistry.

Project description:Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g(1)-value of 2.0090 for the tyrosyl radical was extracted. This g(1)-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν(7a) = 1500 cm(-1)) was found to be insensitive to deuterium-oxide exchange. Additionally, the (18)O-sensitive Fe-O-Fe symmetric stretching (483 cm(-1)) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g(1)-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053-33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.

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Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response.

Publications

Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response.

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