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Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity.

ABSTRACT: The R2 subunit of ribonucleotide reductase from herpes simplex virus type 2 was overproduced with prokaryotic and eukaryotic expression systems. The recombinant R2 purified by a two-step procedure exhibited a 3-fold higher activity when produced in eukaryotic cells. Precise quantification of the R2 concentration at each step of the purification indicated that the activity was not altered during the purification procedure. Moreover, we have observed that the level of R2 expression, in eukaryotic cells as well as in prokaryotic cells, did not influence R2 activity. Extensive characterization of the recombinant R2 purified from eukaryotic and prokaryotic expression systems has shown that both types of pure R2 preparations were similar in their 76 kDa dimer contents (more than 95%) and in their ability to bind the R1 subunit. However, we have found that the higher activity of R2 produced in eukaryotic cells is more probably related to a higher capability of binding the iron cofactor as well as a 3-fold greater ability to generate the tyrosyl free radical.

SUBMITTER: Lamarche N

PROVIDER: S-EPMC1217907 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

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Escherichia coli and herpes-simplex-virus ribonucleotide reductase R2 subunit. Compared reactivities of the redox centres.

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Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase.

Project description:We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.

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The structure of the herpes simplex virus DNA-packaging terminase pUL15 nuclease domain suggests an evolutionary lineage among eukaryotic and prokaryotic viruses.

Project description:Herpes simplex virus 1 (HSV-1), the prototypic member of herpesviruses, employs a virally encoded molecular machine called terminase to package the viral double-stranded DNA (dsDNA) genome into a preformed protein shell. The terminase contains a large subunit that is thought to cleave concatemeric viral DNA during the packaging initiation and completion of each packaging cycle and supply energy to the packaging process via ATP hydrolysis. We have determined the X-ray structure of the C-terminal domain of the terminase large-subunit pUL15 (pUL15C) from HSV-1. The structure shows a fold resembling those of bacteriophage terminases, RNase H, integrases, DNA polymerases, and topoisomerases, with an active site clustered with acidic residues. Docking analysis reveals a DNA-binding surface surrounded by flexible loops, indicating considerable conformational changes upon DNA binding. In vitro assay shows that pUL15C possesses non-sequence-specific, Mg(2+)-dependent nuclease activity. These results suggest that pUL15 uses an RNase H-like, metal ion-mediated catalysis mechanism for cleavage of viral concatemeric DNA. The structure reveals extra structural elements in addition to the RNase H-like fold core and variations in local architecture of the nuclease active site, which are conserved in herpesvirus terminases and bear great similarity to the phage T4 gp17 but are distinct from podovirus and siphovirus orthologs and cellular RNase H, delineating a new evolutionary lineage among a large family of eukaryotic viruses and simple and complex prokaryotic viruses.

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Structures of eukaryotic ribonucleotide reductase I provide insights into dNTP regulation.

Project description:Ribonucleotide reductase catalyzes a crucial step in de novo DNA synthesis and is allosterically controlled by relative levels of dNTPs to maintain a balanced pool of deoxynucleoside triphosphates in the cell. In eukaryotes, the enzyme comprises a heterooligomer of alpha(2) and beta(2) subunits. The alpha subunit, Rnr1, contains catalytic and regulatory sites. Here, we report the only x-ray structures of the eukaryotic alpha subunit of ribonucleotide reductase from Saccharomyces cerevisiae. The structures of the apo-, AMPPNP only-, AMPPNP-CDP-, AMPPNP-UDP-, dGTP-ADP- and TTP-GDP-bound complexes give insight into substrate and effector binding and specificity cross-talk. These are Class I structures with the only fully ordered catalytic sites, including loop 2, a stretch of polypeptide that spans specificity and catalytic sites, conferring specificity. Binding of specificity effector rearranges loop 2; in our structures, this rearrangement moves P294, a residue unique to eukaryotes, out of the catalytic site, accommodating substrate binding. Substrate binding further rearranges loop 2. Cross-talk, by which effector binding regulates substrate preference, occurs largely through R293 and Q288 of loop 2, which are analogous to residues in Thermotoga maritima that mediate cross-talk. However loop-2 conformations and residue-substrate interactions differ substantially between yeast and T. maritima. In most effector-substrate complexes, water molecules help mediate substrate-loop 2 interactions. Finally, the substrate ribose binds with its 3' hydroxyl closer than its 2' hydroxyl to C218 of the catalytic redox pair. We also see a conserved water molecule at the catalytic site in all our structures, near the ribose 2' hydroxyl.

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Cloning and characterization of the R1 and R2 subunits of ribonucleotide reductase from Trypanosoma brucei.

Project description:Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the de novo synthesis of deoxyribonucleotides by directly reducing ribonucleotides to the corresponding deoxyribonucleotides. To keep balanced pools of deoxyribonucleotides, all nonviral RNRs studied so far are allosterically regulated. Most eukaryotes contain a class I RNR, which is a heterodimer of two nonidentical subunits called proteins R1 and R2. We have isolated cDNAs encoding the R1 and R2 proteins from Trypanosoma brucei. The amino acid sequence identities with the mouse R1 and R2 subunits are 58% and 63%, respectively. Recombinant active trypanosome R1 and R2 proteins were expressed in Escherichia coli and purified. The R2 protein contains an iron-tyrosyl free radical center verified by EPR spectroscopy and iron analyses. Measurement of cytidine 5'-diphosphate reduction by the trypanosome RNR in the presence of various allosteric effectors showed that the activity is highest with dTTP, dGTP, or dATP and considerably lower with ATP. The effect of dGTP is either activating (alone) or inhibitory (in the presence of ATP). Filter binding studies indicated that there are two classes of allosteric effector binding sites that bind ATP or dATP (low-affinity dATP site) and ATP, dATP, dGTP, or dTTP (high-affinity dATP site), respectively. Therefore, the structural organization of the allosteric sites is very similar to the mammalian RNRs, whereas the allosteric regulation of cytidine 5'-diphosphate reduction is unique. Hopefully, this difference can be used to target the trypanosome RNR for therapeutic purposes.

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Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis.

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| S-EPMC153024 | biostudies-literature

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

Project description: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.

| S-EPMC2709352 | biostudies-literature

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