The Novel Ribonucleotide Reductase Inhibitor COH29 Inhibits DNA Repair In Vitro
Mei-Chuan ChenBingsen ZhouKeqiang ZhangYate‐Ching YuanFrank UnShuya HuChih‐Ming ChouChun‐Han ChenJun WuYan WangXiyong LiuD. Lynne SmithFrank HongZheng LiuCharles WardenLeila SuLinda H. MalkasYoung‐Min ChungMickey C.‐T. HuYun Yen
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COH29 [N-(4-(3,4-dihydroxyphenyl)-5-phenylthiazol-2-yl)-3,4-dihydroxybenzamide], a novel antimetabolite drug developed at City of Hope Cancer Center, has anticancer activity that stems primarily from the inhibition of human ribonucleotide reductase (RNR). This key enzyme in deoxyribonucleotide biosynthesis is the target of established clinical agents such as hydroxyurea and gemcitabine because of its critical role in DNA replication and repair. Herein we report that BRCA-1–defective human breast cancer cells are more sensitive than wild-type BRCA-1 counterparts to COH29 in vitro and in vivo. Microarray gene expression profiling showed that COH29 reduces the expression of DNA repair pathway genes, suggesting that COH29 interferes with these pathways. It is well established that BRCA1 plays a role in DNA damage repair, especially homologous recombination (HR) repair, to maintain genome integrity. In BRCA1-defective HCC1937 breast cancer cells, COH29 induced more double-strand breaks (DSBs) and DNA-damage response than in HCC1937 + BRCA1 cells. By EJ5– and DR–green fluorescent protein (GFP) reporter assay, we found that COH29 could inhibit nonhomologous end joining (NHEJ) efficiency and that no HR activity was detected in HCC1937 cells, suggesting that repression of the NHEJ repair pathway may be involved in COH29-induced DSBs in BRCA1-deficient HCC1937 cells. Furthermore, we observed an accumulation of nuclear Rad51 foci in COH29-treated HCC1937 + BRCA1 cells, suggesting that BRCA1 plays a crucial role in repairing and recovering drug-induced DNA damage by recruiting Rad51 to damage sites. In summary, we describe here additional biologic effects of the RNR inhibitor COH29 that potentially strengthen its use as an anticancer agent.Keywords:
Ribonucleotide reductase
Ribonucleotide
Ribonucleotide reductase was purified 3400-fold from calf thymus. The enzyme preparation was essentially free of kinases and phosphatases and therefore allowed a conclusive study of the allosteric regulation of a eukaryotic ribonucleotide reductase to be made for the first time. Comparable maximal activities were obtained for the reduction of all four ribonucleotide substrates in the presence of their optimal stimulatory effectors. These and other results strongly argue for the existence of only one ribonucleotide reductase in mammalian cells. No reduction was observed in the absence of effector. The reduction of CDP and UDP both required ATP, with no stimulatory effect of any other nucleoside triphosphate. The only activator of GDP reduction was dTTP and the only activator of ADP reduction was dGTP. Reduction of the purine ribonucleotides was further stimulated by ATP but only in combination with dTTP or dGTP. The reduction of all four ribonucleotides was strongly inhibited by dATP, the inhibition being partly released by ATP. The data can be integrated into a scheme which links ribonucleotide reduction to DNA synthesis.
Ribonucleotide reductase
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Purine metabolism
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Deoxyribonucleotides
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Mycobacterium smegmatis mc2 155 contains a Ribonucleotide reductases (RNR), which catalyses the irreversible reduction of ribonucleotides to the corresponding 2 Ìdeoxyribonucleotides required for DNA replication and cell proliferation. The aim of this work was the development of a rapid assay for ribonucleotide reduction by Mycobacterium smegmatis mc 2 155 and their biochemical characterisation. For this purpose, the cells of Mycobacterium smegmatis mc 2 155 were permeabilized with two organic solvents toluene and ether for two times (2,5 min) to develop a new assay for ribonucleotide reduction. Due the importance of the growth phase in determining the yield of biomass and ribonucleotide reductase activity of Mycobacterium smegmatis mc 2 155, a correlation between Ribonucleotide reductase activity and growth of Mycobacterium smegmatis mc 2 155 in modified seed medium has been investigated. For the enrichment of the Ribonucleotide reductase, different purification procedure has been achieved by using fast protein liquid chromatography (FPLC) with superdex G-200 chromatography and Phenyl-Superose HR 5/5 and the enzyme activity was assayed by using (HPLC). Ribonucleotide reductase activity was detectable in the 40-60% ammonium sulphate fraction. A further purification procedure by gel filtration on the superdex G-200 led to a dissociation of the both subunits. Therefore, a biochemical complementation assay was necessary to identify ribonucleotide reductase activity. The obtained specific activity of the purified protein was 1790 pmol per mg per min with an overall yield of 10%. The purified small subunit of MS2- protein was detected on SDS-PAGE, which was showed a strong band that corresponds to an apparent molecular weight of 38.5 KDa. The obtained results of ribonuleotide reduction activity with ether permeabilized cells of Mycobacterium smegmatis mc 2 155 presented a comparable enzyme activity for both times, while with toluene permeabilized cells indicated a low enzyme activity. Furthermore, the obtained results of the correlation between ribonucleotide reductase activity and the growth showed that Ribonucleotide redutase is a peak enzyme. Finally, the permeabilisation of the cells of M. smegmatis mc 2 155 with ether and toluene for short time facilitated us to develop a rapid assay for ribonucleotide reductase activity of others gram positive bacteria.
Ribonucleotide reductase
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Ribonucleotide
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Ribonucleotide reduction, the unique step in the pathway to DNA synthesis, is catalyzed by enzymes via radical‐dependent redox chemistry involving an array of diverse metallocofactors. The nucleotide reduction gene ( nrdF ) encoding the metallocofactor containing small subunit (R2F) of the Corynebacterium ammoniagenes ribonucleotide reductase was reintroduced into strain C. ammoniagenes ATCC 6872. Efficient homologous expression from plasmid pOCA2 using the tac ‐promotor enabled purification of R2F to homogeneity. The chromatographic protocol provided native R2F with a high ratio of manganese to iron (30 : 1), high activity (69 μmol 2′‐deoxyribonucleotide·mg −1 ·min −1 ) and distinct absorption at 408 nm, characteristic of a tyrosyl radical (Y˙), which is sensitive to the radical scavenger hydroxyurea. A novel enzyme assay revealed the direct involvement of Y˙ in ribonucleotide reduction because 0.2 nmol 2′‐deoxyribonucleotide was formed, driven by 0.4 nmol Y˙ located on R2F. X‐band electron paramagnetic resonance spectroscopy demonstrated a tyrosyl radical at an effective g ‐value of 2.004. Temperature dependent X/Q‐band EPR studies revealed that this radical is coupled to a metallocofactor. Similarities of the native C. ammoniagenes ribonucleotide reductase to the in vitro activated Escherichia coli class Ib enzyme containing a dimanganese(III)‐tyrosyl metallocofactor are discussed.
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Ribonucleotide reductases (RNRs) use radical-based chemistry to catalyze the conversion of all four ribonucleotides to deoxyribonucleotides. The ubiquitous nature of RNRs necessitates multiple RNR classes that differ from each other in terms of the phosphorylation state of the ribonucleotide substrates, oxygen tolerance, and the nature of both the metallocofactor employed and the reducing systems. Although these differences allow RNRs to produce deoxyribonucleotides needed for DNA biosynthesis under a wide range of environmental conditions, they also present a challenge for establishment of a universal activity assay. Additionally, many current RNR assays are limited in that they only follow the conversion of one ribonucleotide substrate at a time, but in the cell, all four ribonucleotides are actively being converted into deoxyribonucleotide products as dictated by the cellular concentrations of allosteric specificity effectors. Here, we present a liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based assay that can determine the activity of both aerobic and anaerobic RNRs on any combination of substrates using any combination of allosteric effectors. We demonstrate that this assay generates activity data similar to past published results with the canonical Escherichia coli aerobic class Ia RNR. We also show that this assay can be used for an anaerobic class III RNR that employs formate as the reductant, i.e. Streptococcus thermophilus RNR. We further show that this class III RNR is allosterically regulated by dATP and ATP. Lastly, we present activity data for the simultaneous reduction of all four ribonucleotide substrates by the E . coli class Ia RNR under various combinations of allosteric specificity effectors. This validated LC-MS/MS assay is higher throughput and more versatile than the historically established radioactive activity and coupled RNR activity assays as well as a number of the published HPLC-based assays. The presented assay will allow for the study of a wide range of RNR enzymes under a wide range of conditions, facilitating the study of previously uncharacterized RNRs.
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Deoxyribonucleotides
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