Large Subunit Of Ribonucleotide Reductase Research Articles

Stress up-regulates neuronal expression of the herpes simplex virus type 2 large subunit of ribonucleotide reductase (R1; ICP10) by activating activator protein 1

Herpes simplex virus type 2 (HSV-2) genes expressed in neuronal cells in response to stress stimuli that trigger latency reactivation are largely unknown. Using a chloramphenicol acetyltransferase (CAT) reporter assay we found that stress caused a significant (P < .001) increase in ICP10 expression in neuronal cells. Up-regulation correlated with activator protein (AP)-1 activation, notably c-Jun and c-Fos that bind cognate elements in the ICP10 promoter. It was blocked by mutation of the AP-1 motifs in the ICP10 promoter. ICP10 expression protected neuronal cells from stress-induced apoptosis. The data suggest that ICP10 may contribute to HSV-2 reactivation by increasing neuronal survival.

An increase in the expression of ribonucleotide reductase large subunit 1 is associated with gemcitabine resistance in non-small cell lung cancer cell lines.

The mechanisms of resistance to the antimetabolite gemcitabine in non-small cell lung cancer have not been extensively evaluated. In this study, we report the generation of two gemcitabine-selected non-small cell lung cancer cell lines, H358-G200 and H460-G400. Expression profiling results indicated that there was evidence for changes in the expression of 134 genes in H358-G200 cells compared with its parental line, whereas H460-G400 cells exhibited 233 genes that appeared to be under- or overexpressed compared with H460 cells. However, only the increased expression of ribonucleotide reductase subunit 1 (RRM1), which appeared in both resistant cell lines, met predefined analysis criteria for genes to investigate further. Quantitative PCR analysis demonstrated H358-G200 cells had a greater than 125-fold increase in RRM1 RNA expression. Western blot analysis confirmed high levels of RRM1 protein in this line compared with the gemcitabine-sensitive parent. No significant change in the expression of RRM2 was observed in either cell line, although both gemcitabine-resistant cell lines had an approximate 3-fold increase in p53R2 protein. A partial revertant of H358-G200 cells had reduced levels of RRM1 protein (compared with G200 cells), without observed changes in RRM2 or p53R2. In vitro analyses of ribonucleotide reductase activity demonstrated that despite high levels of RRM1 protein, ribonucleotide reductase activity was not increased in H358-G200 cells when compared with parental cells. The cDNA encoding RRM1 from H358-G200 cells was cloned and sequenced but did not reveal the presence of any mutations. The results from this study indicate that the level of RRM1 may affect gemcitabine response. Furthermore, RRM1 may serve as a biomarker for gemcitabine response.

1031. Improved Anti-Brain Tumor Efficacy of Δγ134.5 HSV Expressing Cytosine Deaminase

Δγ134.5 Herpes Simplex Viruses (HSV) are non-neurovirulent genetic constructs that have been shown to grow conditionally in a wide variety of mouse and human tumor cells in vitro. Despite the fact that these replication competent HSV can readily kill susceptible tumor cells in vitro, intratumoral (i.t.) injection of these viruses in animals bearing intracranial (i.c.) or flank tumors has not uniformly inhibited tumor growth. To improve in vivo tumoricidal activity, we have inserted the bacterial gene for cytosine deaminase (CD) in the loci from which the γ134.5 genes responsible for neurovirulence were deleted. This initial virus was designated HSV M012. CD efficiently converts the relatively non-toxic prodrug 5-fluorocytosine (5-FC) to the potently toxic 5-fluorouracil (5-FU) anti-metabolite. CD has been used in a wide variety of experimental and clinical gene therapy studies to eliminate tumor cells by impairing pyrimidine synthesis in both RNA and DNA pathways. Moreover, 5-FU has been proven to be a true radiation sensitizer, which offers the possibility of using combined modalities to eliminate tumors. A second HSV construct was made, M1012, which also carried an insertional deletion of the virus gene encoding for the large subunit of ribonucleotide reductase. The rationale was that virus-produced ribonucleotide reductase in infected cells may be able to incorporate 5-(F)UMP into virus RNA synthesis and impair virus replication. Both viruses were shown to infect and kill human tumor cells and produce detectable amounts of bioactive CD. Conversion of 5-FC to 5-FU by infected human glioma cells was quantified. In vitro, mouse Neuro-2A (N2A) cells are relatively resistant to killing by HSV at multiplicities of infection (MOI) ranging from 1 to 20. Addition of increasing concentrations of 5-FC (100-1,000 μM) resulted in significant killing of N2A cells at progressively lower MOI. C-b.17 scid mice bearing i.c. gliomas induced by injection of 5×105 U87MG human glioma cells were treated 7 days later by i.t. injection of 107 pfu of either HSV M012 or HSV M1012 or received an equivalent volume of saline. Two days later mice began a 7-day regimen of twice daily i.p. injections of 500 mg/kg 5-FC, then followed for survival. Mice receiving saline i.t. and 5-FC had a median survival time (MST) of 34 days, characteristic of this model. Those that received M012 i.t. followed by saline (MST = 43 days) or 5-FC (MST = 55 days) had significantly enhanced survivals (over saline +5-FC) but there were no long-term survivors. Likewise mice receiving M1012 i.t. followed by saline experienced increased survival (MST=53 days) but without any cures. However, mice receiving M1012 followed by 5-FC have yet to reach a median survival with over 66% long-term survivors at 210 days. These and other data not described suggest that the addition of prodrug therapy to virus-mediated oncolysis may afford a significant improvement over virus therapy alone. Further, this study suggests that HSV ribonucleotide reductase may exert a negative survival effect on the viral vector, limiting its potential efficacy as a gene therapy agent.

ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana.

Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation, transmitting DNA damage signals to downstream effectors of cell-cycle progression. In animals, ATR is an essential gene. Here, we find that Arabidopsis (Arabidopsis thaliana) atr-/- mutants were viable, fertile, and phenotypically wild-type in the absence of exogenous DNA damaging agents but exhibit altered expression of AtRNR1 (ribonucleotide reductase large subunit) and alteration of some damage-induced cell-cycle checkpoints. atr mutants were hypersensitive to hydroxyurea (HU), aphidicolin, and UV-B light but only mildly sensitive to gamma-radiation. G2 arrest was observed in response to gamma-irradiation in both wild-type and atr plants, albeit with slightly different kinetics, suggesting that ATR plays a secondary role in response to double-strand breaks. G2 arrest also was observed in wild-type plants in response to aphidicolin but was defective in atr mutants, resulting in compaction of nuclei and subsequent cell death. By contrast, HU-treated wild-type and atr plants arrested in G1 and showed no obvious signs of cell death. We propose that, in plants, HU invokes a novel checkpoint responsive to low levels of deoxynucleotide triphosphates. These results demonstrate the important role of cell-cycle checkpoints in the ability of plant cells to sense and cope with problems associated with DNA replication.

UV-C response of the ribonucleotide reductase large subunit involves both E2F-mediated gene transcriptional regulation and protein subcellular relocalization in tobacco cells.

E2F factors are implicated in various cellular processes including specific gene induction at the G1/S transition of the cell cycle. We present in this study a novel regulatory aspect for the tobacco large subunit of ribonucleotide reductase (R1a) and its encoding gene (RNR1a) in the UV-C response. By structural analyses, two E2F sites were identified on the promoter of this gene. Functional analysis showed that, in addition to their role in the specific G1/S induction of the RNR1a gene, both E2F sites were important for regulating specific RNR1a gene expression in response to UV-C irradiation in non-synchronized and synchronized cells. Concomitantly, western blot and cellular analyses showed an increase of a 60 kDa E2F factor and a transient translocation of a GFP-R1a protein fusion from cytoplasm to nucleus in response to UV irradiation.

Gene content and density in banana (Musa acuminata) as revealed by genomic sequencing of BAC clones

The complete sequence of Musa acuminata bacterial artificial chromosome (BAC) clones is presented and, consequently, the first analysis of the banana genome organization. One clone (MuH9) is 82,723 bp long with an overall G+C content of 38.2%. Twelve putative protein-coding sequences were identified, representing a gene density of one per 6.9 kb, which is slightly less than that previously reported for Arabidopsis but similar to rice. One coding sequence was identified as a partial M. acuminata malate synthase, while the remaining sequences showed a similarity to predicted or hypothetical proteins identified in genome sequence data. A second BAC clone (MuG9) is 73,268 bp long with an overall G+C content of 38.5%. Only seven putative coding regions were discovered, representing a gene density of only one gene per 10.5 kb, which is strikingly lower than that of the first BAC. One coding sequence showed significant homology to the soybean ribonucleotide reductase (large subunit). A transition point between coding regions and repeated sequences was found at approximately 45 kb, separating the coding upstream BAC end from its downstream end that mainly contained transposon-like sequences and regions similar to known repetitive sequences of M. acuminata. This gene organization resembles Gramineae genome sequences, where genes are clustered in gene-rich regions separated by gene-poor DNA containing abundant transposons.

Evaluation of the antitumor effects of Herpes simplex virus lacking ribonucleotide reductase in a murine retinoblastoma model

Purpose. To determine if an attenuated herpes simplex virus (HSV) lacking the large subunit of ribonucleotide reductase has antitumor effects in a transgenic mouse model of retinoblastoma (LHβTAg).Methods. LHβTAg mice were injected ocularly with 1 × 106 pfu of the hrR3 virus and tumor sizes were measured 3 weeks later. Replication of the virus in the eye and cultured murine retinoblastoma cells was tested by titration. Distribution of the virus in tumor was measured by X-gal staining.Results. Intraocular injection of mice with hrR3 (n = 24) did not result in a significant reduction in tumor size compared to uninjected (n = 24) or PBS injected controls (n = 16). Neither the hrR3, nor the HSV RE6 mutant, which was previously shown to have antitumor effects in vivo, replicated in cultured murine tumor cells in vitro, compared to wild-type HSV. The hrR3 virus also did not replicate significantly in tumor cells in vivo, compared to normal eye tissue.Conclusions. These results suggest that mutant HSV lacking ribonucleotide reductase do not display oncolytic activity in the LHβTAg mouse and that this model may not be suitable for studying viral oncolysis as a therapy for retinoblastoma.

MCB-mediated regulation of cell cycle-specific cdc22+ transcription in fission yeast.

The cdc22+ gene of the fission yeast, Schizosaccharomyces pombe, encodes the large subunit of ribonucleotide reductase, and is periodically expressed during the mitotic cell cycle, transcript abundance reaching a maximum at the G1-S boundary. This regulation of expression is controlled by a transcription factor complex called DSC1, which binds to MCB motifs (ACGCGT) present in the promoter of cdc22+. cdc22+ has a complex pattern of MCBs, including two clusters of four motifs each, one of which is located within the transcribed region. We show that both clusters of MCBs contribute to the regulation of cdc22+ expression during the cell cycle, each having a different role. The MCB cluster within the transcribed region has the major role in regulating cdc22+, as its removal results in loss of transcription. The upstream cluster, instead, controls cell cycle-specific transcription through a negative function, as its removal results in expression of cdc22+ throughout the cell cycle. Both MCB clusters bind DSC1. We show that the interaction of DSC1 with the MCB cluster within the transcribed region has a high "on-off" rate, suggesting a mechanism by which DSC1 could activate expression, and still allow RNA polymerase to pass during transcription. Finally, we show that both clusters are orientation-dependent in their function. The significance of these results, in the context of MCB-mediated regulation of G1-S expression in fission yeast, is discussed.

Transcriptional analysis of the white spot syndrome virus major virion protein genes.

White spot syndrome virus (WSSV) is a member of a new virus family (Nimaviridae) infecting crustaceans. The regulation of transcription of WSSV genes is largely unknown. Transcription of the major WSSV structural virion protein genes, vp28, vp26, vp24, vp19 and vp15, was studied to search for common promoter motifs for coordinate expression. The temporal expression of these genes and both 5' and 3' ends of the mRNA were determined, using infected crayfish gill tissue as a RNA source. RT-PCR showed that all five genes are expressed late in infection compared to the early ribonucleotide reductase large subunit gene. 5' RACE studies revealed a consensus late transcription initiation motif for only two of the five major virion protein genes. This motif was only found in one other upstream region of the putative translational start site of a gene with unknown function (ORF 158). No other conserved sequence motifs could be detected in the sequences surrounding the transcriptional start sites of the five major virion protein genes. All 5' ends were located about 25 nt downstream of an A/T rich sequence, including the consensus TATA-box sequence for vp15. The absence of a consensus motif is distinct from gene regulation of other large dsDNA viruses and suggests a unique regulation of WSSV transcription, in line with its unique taxonomic position.

Analysis of the complete genome sequence of the Hz-1 virus suggests that it is related to members of the Baculoviridae.

We report the complete sequence of a large rod-shaped DNA virus, called the Hz-1 virus. This virus persistently infects the Heliothis zea cell lines. The Hz-1 virus has a double-stranded circular DNA genome of 228,089 bp encoding 154 open reading frames (ORFs) and also expresses a persistence-associated transcript 1, PAT1. The G+C content of the Hz-1 virus genome is 41.8%, with a gene density of one gene per 1.47 kb. Sequence analysis revealed that a 9.6-kb region at 43.6 to 47.8 map units harbors five cellular genes encoding proteins with homology to dUTP pyrophosphatase, matrix metalloproteinase, deoxynucleoside kinase, glycine hydroxymethyltransferase, and ribonucleotide reductase large subunit. Other cellular homologs were also detected dispersed in the viral genome. Several baculovirus homologs were detected in the Hz-1 virus genome. These include PxOrf-70, PxOrf-29, AcOrf-81, AcOrf-96, AcOrf-22, VLF-1, RNA polymerase LEF-8 (orf50), and two structural proteins, p74 and p91. The Hz-1 virus p74 homolog shows high structural conservation with a double transmembrane domain at its C terminus. Phylogenetic analysis of the p74 revealed that the Hz-1 virus is evolutionarily distant from the baculoviruses. Another distinctive feature of the Hz-1 virus genome is a gene that is involved in insect development. However, the remainder of the ORFs (81%) encoded proteins that bear no homology to any known proteins. In conclusion, the sequence differences between the Hz-1 virus and the baculoviruses outnumber the similarities and suggest that the Hz-1 virus may form a new family of viruses distantly related to the Baculoviridae:

Development of HSV-specific CD4+ Th1 responses and CD8+ cytotoxic T lymphocytes with antiviral activity by vaccination with the HSV-2 mutant ICP10ΔPK

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10ΔPK) protects from HSV-2 challenge in the mouse and guinea pig cutaneous and vaginal models and reduces the incidence and frequency of recurrent disease (Vaccine (17) (1999) 1951; Vaccine (19) (2001) 1879). The present studies were designed to identify the immune responses induced by ICP10ΔPK and define the component responsible for protective activity. We found that ICP10ΔPK elicits a predominant HSV-specific T helper type 1 (Th1) response, as evidenced by: (1) higher levels of HSV-specific IgG2a (Th1) than IgG1 (Th2) isotypes and (2) higher numbers of CD4+ IFN-γ than IL-10 secreting T cells in popliteal lymph nodes. This Th1 response pattern was associated with a significant increase in the levels of IL-12 produced by dendritic cells from ICP10ΔPK than HSV-2 immunized animals. Lymph node cells (LNCs) from ICP10ΔPK immunized mice had significantly higher levels of HSV-2 specific cytolytic activity than LNCs from mice immunized with HSV-2 and it was mediated by CD8+ T cells. CD8+ CTL were not seen in LNCs from HSV-2 immunized mice. In adoptive transfer experiments, CD8+ T cells and, to a lower extent, CD4+ T cells from ICP10ΔPK immunized mice inhibited HSV-2 replication, suggesting that they are involved in the protective immunity induced by ICP10ΔPK vaccination.

Characterization of Monomeric and Dimeric Forms of Recombinant Sml1p-histag Protein by Electrospray Mass Spectrometry

Sml1p is small protein that binds to and inhibits the activity of ribonucleotide reductase (RNR)33Abbreviations used: RNR, ribonucleotide reductase; ORF, open reading frame; ES-FTICR-MS, electrospray Fourier transform ion cyclotron resonance mass spectrometry; NTA, nitrilotriacetic acid; IOD, image optical density; SORI-CAD, sustained off-resonance irradiation collision-activated dissociation; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; DTT, dithiothreitol., a protein enzyme complex that controls the balance and level of the cellular deoxynucleotide diphosphate pools that are critical for DNA synthesis and repair. In this respect, Sml1p is a checkpoint protein whose function is to regulate the activity of the large subunit of RNR (Rnr1p). Sml1p is thought to be regulated by the MEC1/RAD53 cell cycle checkpoint pathway. Neither the structure of Sml1p nor its complex to Rnr1p is well known. In this report, we describe how a recombinant Sml1p-histag protein (in both monomeric and dimeric forms) can be characterized with electrospray mass spectrometry. Mass spectrometry can play a vital role in the study of the Sml1p-Rnr1p complex by: (1) confirming the identities and purities of recombinant proteins such as Sm1lp-histag (with mass accuracy and resolution far superior to SDS–PAGE) and (2) verifying the presence or absence of PTM, chemical modifications, or metal–ion binding to the protein species, which may alter the function and binding of the protein partners.

Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells.

Recently, a homologue of the small subunit of mammalian ribonucleotide reductase (RNR) was discovered, called p53R2. Unlike the well characterized S phase-specific RNR R2 protein, the new form was induced in response to DNA damage by the p53 protein. Because the R2 protein is specifically degraded in late mitosis and absent in G0/G1 cells, the induction of the p53R2 protein may explain how resting cells can obtain deoxyribonucleotides for DNA repair. However, no direct demonstration of RNR activity of the p53R2 protein was presented and furthermore, no corresponding RNR large subunit was identified. In this study we show that recombinant, highly purified human and mouse p53R2 proteins contain an iron-tyrosyl free radical center, and both proteins form an active RNR complex with the human and mouse R1 proteins. UV irradiation of serum-starved, G0/G1-enriched mouse fibroblasts, stably transformed with an R1 promoter-luciferase reporter gene construct, caused a 3-fold increase in luciferase activity 24 h after irradiation, paralleled by an increase in the levels of R1 protein. Taken together, our data indicate that the R1 protein can function as the normal partner of the p53R2 protein and that an R1-p53R2 complex can supply resting cells with deoxyribonucleotides for DNA repair.

Reverse transcriptase template switching during reverse transcriptase–polymerase chain reaction: Artificial generation of deletions in ribonucleotide reductase mRNA

Using reverse transcriptase–polymerase chain reaction (RT-PCR), we have recently described a bona fide deletion within the coding sequence of the large subunit of ribonucleotide reductase (R1) mRNA in colon cancer. Consecutive studies have raised questions about the nature of this phenomenon, because the corresponding genomic alteration at the DNA level or an aberrant protein could not be detected. Thus we considered an in vitro artifact during RT-PCR as a possible explanation for this observation. In contrast to reverse transcriptase, Taq DNA polymerase or C. therm DNA polymerase did not generate the aberrant product, suggesting the demand for the template switching activity intrinsic to retroviral reverse transcriptases. In fact, virtually the same deletion was observed in RT-PCR experiments when in vitro transcribed R1 mRNA was used. Considering structural prerequisites for template switching within R1 mRNA, we show that two direct repeats adjacent to a strong stem-loop secondary structure flank the deleted region of 1851 base pairs. Because several mRNAs encoding proteins of clinical and diagnostic importance fulfill these criteria, template switching enhances the potential risk of observing artifacts when interpreting results from RT-PCR studies. As shown in the present example, this may involve the artificial generation and the misinterpretation of PCR fragments amplified from targets relevant to tumor biology or cancer pharmacology. As a possible solution, one-step PCR with C. therm polymerase should be considered. This polymerase eliminates the artificial generation of aberrant mRNA signals observed during cDNA synthesis. (J Lab Clin Med 2001;137:422-8)

Sequencing and amplified restriction fragment length polymorphism analysis of ribonucleotide reductase large subunit gene of the white spot syndrome virus in blue crab (Callinectes sapidus) from American Coastal Waters.

In the present study, the existence of white spot syndrome virus (WSSV) in blue crab (Callinectes sapidus) collected from 3 different American coastal waters (New York, New Jersey, and Texas) was confirmed by 2-step diagnostic polymerase chain reaction and in situ hybridization analysis. When geographic isolates were also compared using a gene that encodes the WSSV ribonucleotide reductase large subunit RR1 (WSSV rr1), a C(1661)-to-T point mutation was found in the New Jersey WSSV isolated. This point mutation, which resulted in the creation of an additional RsaI endonuclease recognition site, was not found in the WSSV from the New York and Texas blue crab samples, or in the WSSV Taiwan isolate, or in any of the other WSSV geographical isolates for which data are available. WSSV rr1-specific RsaI amplified restriction fragment length polymorphism of an amplified 1156-bp fragment thus distinguished the New Jersey blue crab samples from the other WSSV isolates.

A growth and latency compromised herpes simplex virus type 2 mutant (ICP10ΔPK) has prophylactic and therapeutic protective activity in guinea pigs

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10ΔPK) protects from fatal HSV-2 challenge in the mouse model (Aurelian L, Kokuba H, Smith CC. Vaccine potential of a Herpes Simplex Virus type 2 mutant deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10). Vaccine 1999;17:1951–1963). Here we report the results of our studies with ICP10ΔPK in the guinea pig model of recurrent HSV-2 disease. ICP10ΔPK was also compromised for growth and disease causation in this model. It was not isolated from latently infected ganglia by explant co-cultivation. The proportions of latently infected ganglia were significantly lower for ICP10ΔPK than HSV-2 [3/25 (12%) and 7/10 (70%), respectively]. Similar results were obtained for the levels of viral DNA (8×10 3 and 2×10 5 molecules/ganglion for ICP10ΔPK and HSV-2, respectively]. ICP10ΔPK immunization caused a significant ( P≤0.001) decrease in the proportion of animals with primary [1/14 (6%) and 16/16 (100%) for ICP10ΔPK and PBS, respectively) and recurrent [1/14 (6%) and 11/14 (79%) for ICP10ΔPK and PBS, respectively) HSV-2 skin lesions. It also protected from genital HSV-2 disease [1/10 and 10/10 for ICP10ΔPK and PBS, respectively] and decreased the severity of the lesions in both models. Quantitative PCR (Q-PCR) with primers that distinguish between HSV-2 and ICP10ΔPK indicated that immunization reduced the proportion of ganglia positive for HSV-2 DNA [8/25 (32%) and 7/10 (70%) for ICP10ΔPK and PBS, respectively) and its levels [3×10 3 and 2×10 5 molecules/ganglion for ICP10ΔPK and PBS, respectively]. The proportion of HSV-2 infected animals with recurrent disease was also significantly ( P≤0.001) decreased by immunization with ICP10ΔPK [1/15 (7%) and 11/14 (79%) with recurrent disease for ICP10ΔPK and PBS, respectively], suggesting that ICP10ΔPK has prophylactic and therapeutic activity in the guinea pig.

A Novel Gene Expressed in Human Keratinocytes with Long-Term In Vitro Growth Potential is Required for Cell Growth

The herpes simplex virus large subunit of ribonucleotide reductase differs from its counterparts in eukaryotic and prokaryotic cells and in other viruses in that it contains a unique domain that codes for a distinct serine-threonine protein kinase that activates the Ras/MEK/MAPK mitogenic pathway and is required for virus growth. Previous studies suggested that ribonucleotide reductase protein kinase was co-opted from a cellular gene. Cellular genes similar to ribonucleotide reductase protein kinase were not cloned, however, and their function is unknown. Here we report that a novel gene (H11) that codes for a protein similar to herpes simplex virus 2 ribonucleotide reductase protein kinase, is expressed in skin tissues, cultured keratinocytes, and the keratinocyte cell line A431. The protein is phosphorylated and it associates with the plasma membrane. H11 is expressed in keratinocytes with long-term in vitro growth potential and is coexpressed with high levels of adhesion molecules involved in signal transduction, such as beta1 integrin. Antisense oligonucleotides that inhibit H11 expression inhibit DNA synthesis and keratinocyte proliferation, suggesting that H11 expression is required for cell growth.

Mutational and structural analyses of the ribonucleotide reductase inhibitor Sml1 define its Rnr1 interaction domain whose inactivation allows suppression of mec1 and rad53 lethality.

In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1 or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53 inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53 strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.

Identification of ribonucleotide reductase protein R1 as an activator of microtubule nucleation in Xenopus egg mitotic extracts.

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

Transcriptional Analysis of the Ribonucleotide Reductase Genes of Shrimp White Spot Syndrome Virus

The causative agent of white spot syndrome (WSS) is a large double-stranded DNA virus, WSSV, which is probably a representative of a new genus, provisionally called Whispovirus. From previously constructed WSSV genomic libraries of a Taiwan WSSV isolate, clones with open reading frames (ORFs) that encode proteins with significant homology to the class I ribonucleotide reductase large (RR1) and small (RR2) subunits were identified. WSSV rr1 and rr2 potentially encode 848 and 413 amino acids, respectively. RNA was isolated from WSSV-infected shrimp at different times after infection and Northern blot analysis with rr1- and rr2-specific riboprobes found major transcripts of 2.8 and 1.4 kb, respectively. 5′ RACE showed that the major rr1 transcript started at a position of −84 (C) relative to the ATG translational start, while transcription of the rr2 gene started at nucleotide residue −68 (T). A consensus motif containing the transcriptional start sites for rr1 and rr2 was observed (TCAc/tTC). Northern blotting and RT-PCR showed that the transcription of rr1 and rr2 started 4–6 h after infection and continued for at least 60 h. The rr1 and rr2 genes thus appear to be WSSV “early genes.”