Adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate: A simultaneous protein binding assay

The adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate contents of microliter quantities of urine can be determined simultaneously by combining individual protein binding assays for the two nucleotides. ³²P-labeled adenosine 3′,5′-monophosphate is bound to a protein from bovine skeletal muscle, while a lobster muscle protein preparation is utilized for binding of ³H-labeled guanosine 3′,5′-monophosphate.     

Coordination properties of 3′,5′-cyclic adenosine monophosphate towards copper(II) ions

The metal binding ability of 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) molecule using copper(II) ion, as an example of biologically available divalent metal ion, was investigated by potentiometry, EPR and differential spectroscopy (UV-Vis, CD). One complex with stoichiometry Cu(3′,5′-cAMP)⁺ was found, where Cu(II) ion is bound by N-7 nitrogen of adenine moiety.     

Binding of 2′-Amino-2′-Deoxycytidine-5′-Triphosphate to Norovirus Polymerase Induces Rearrangement of the Active Site

Crystal structures of a genogroup II.4 human norovirus polymerase bound to an RNA primer-template duplex and the substrate analogue 2′-amino-2′-deoxycytidine-5′-triphosphate have been determined to 1.8 Å resolution. The alteration of the substrate-binding site that is required to accommodate the 2′-amino group leads to a rearrangement of the polymerase active site and a disruption of the coordination shells of the active-site metal ions. The mode of binding seen for 2′-amino-2′-deoxycytidine-5′-triphosphate suggests a novel molecular mechanism of inhibition that may be exploited for the design of inhibitors targeting viral RNA polymerases.     

Adenosine-5′-phosphosulfate kinase is essential for Arabidopsis viability

In Arabidopsis thaliana, adenosine-5′-phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability.     

Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences ofLabeo rohita, Cirrhina mrigala andCatla catla have been cloned, characterized and overexpressed inEscherichia coli. These sequences show 96–98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.     

2′-5′-Oligoadenylate synthetase is activated by a specific RNA sequence motif

2′-5′-Oligoadenylate synthetase plays a central role in the cellular innate antiviral response. Although activation of 2′-5′-oligoadenylate synthetase by double stranded RNA was discovered more than 30 years ago it is still unclear which sequence features are required by an RNA to activate the enzyme. A pool of chemically synthesized short double stranded RNAs of specific sequence was used to probe 2′-5′-oligoadenylate synthetase activation. It was found that activating double stranded RNAs contain the following motif: NNWWNNNNNNNNNWGN. Verification of this sequence motif in a pool of 102 small double stranded RNAs demonstrated a false positive prediction rate of 8% and a false negative prediction rate of 12%. The sequence motif identified provides mechanistic insight into the mechanism of 2′-5′-oligoadenylate synthetase activation by double stranded RNA and allows theoretical predictions whether a given RNA molecule has the capability to activate 2′-5′-oligoadenylate synthetase.     

A heterodimer of human 3′-phospho-adenosine-5′-phosphosulphate (PAPS) synthases is a new sulphate activating complex

3′-Phospho-adenosine-5′-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Förster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5′-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.     

The X-ray crystal structure of APR-B,an atypical adenosine 5′-phosphosulfate reductase from Physcomitrella patens

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe₄S₄] cluster, where adenosine 5′-phosphosulfate (APS) reductases possess a cluster and 3′-phosphoadenosine 5′-phosphosulfate reductases do not. The exception is the APR-B isoform of APS reductase from the moss Physcomitrella patens, which lacks a cluster. The crystal structure of APR-B, the first for a plant sulfonucleotide reductase, is consistent with a preference for APS. Structural conservation with bacterial APS reductase rules out a structural role for the cluster, but supports the contention that it enhances the activity of conventional APS reductases.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

PRMT5 is essential for the eIF4E-mediated 5′-cap dependent translation

It is becoming clear that PRMT5 plays essential roles in cell cycle progression, survival, and responses to external stresses. However, the precise mechanisms underlying such roles of PRMT5 have not been clearly understood. Previously, we have demonstrated that PRMT5 participates in cellular adaptation to hypoxia by ensuring 5′-cap dependent translation of HIF-1α. Given that c-Myc and cyclin D1 expressions are also tightly regulated in 5′-cap dependent manner, we here tested the possibility that PRMT5 promotes cell proliferation by increasing de novo syntheses of the oncoproteins. c-Myc and cyclin D1 were found to be noticeably downregulated by PRMT5 knock-down. A RNA immunoprecipitation analysis, which can identify RNA–protein interactions, showed that PRMT5 is required for the interaction among eIF4E and 5′-UTRs of HIF-1α, c-Myc and cyclin D1 mRNAs. In addition, PRMT5 knock-down inhibited cell proliferation by inducing cell cycle arrest at the G1 phase. More importantly, ectopic expression of eIF4E significantly rescued the cell cycle progression and cell proliferation even in PRMT5-deficeint condition. Based on these results, we propose that PRMT5 determines cell fate by regulating 5′-cap dependent translation of proteins essential for proliferation and survival.     

Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83) in human plasma

An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2′,3′-dideoxy-3′-fluorouridine (935U83; I) directly and its 5′-glucuronide metabolite (5-chloro-2′,3′-dideoxy-5′-O-β- -glucopyranuronosyl-3′-fluorouridine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with β-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from −5.5 to 7.1% during the validation and from −6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-μl sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.     

Repair efficiency of (5′S)-8,5′-cyclo-2′-deoxyguanosine and (5′S)-8,5′-cyclo-2′-deoxyadenosine depends on the complementary base

5′-R and 5′-S diastereoisomers of 8,5′-cyclo-2′-deoxyadenosine (cdA) and 8,5′-cyclo-2′-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24–32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N²-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N²-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.     

The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3′-5′ exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3′-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3′ penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3′-5′ exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3′-penultimate deoxyinosines derived from endo V nicking reaction.     

RecA-mediated multistrand formation for cloning PCR products into vectors: simplified process for 5'-rapid amplification of cDNA ends

I have developed a novel rapid amplification of cDNA ends (RACE) technology that uses multistranded DNA formation mediated by the RecA protein. Multistranded DNA can readily be formed at the terminus of double-stranded DNA by a complementary single-stranded DNA in the presence of RecA and exonuclease I. The possibility of applying this finding to the direct cloning of a 5'-RACE product onto a cDNA fragment, which does not require the use of restriction endonucleases, was explored. The results show that the terminal multistranded structure formed by the RecA-mediated reaction can be applied to RACE systems. Modifications to the RACE protocol to improve the effectiveness of the technique are also suggested.     

Identification and quantification of (5′R)- and (5′S)-8,5′-cyclo-2′-deoxyadenosines in human urine as putative biomarkers of oxidatively induced damage to DNA

Biomarkers of oxidatively induced DNA damage are of great interest and can potentially be used for the early detection of disease, monitoring the progression of disease and determining the efficacy of therapy. The present work deals with the measurement in human urine of (5′R)-8,5′-cyclo-2′-deoxyadenosine (R-cdA) and (5′S)-8,5′-cyclo-2′-deoxyadenosine (S-cdA). These modified nucleosides had hitherto not been considered or investigated to be present in urine as possible biomarkers of oxidatively induced DNA damage. Urine samples were collected from volunteers, purified and analyzed by LC-MS/MS with isotope-dilution. R-cdA and S-cdA were detected in urine and quantified. Creatinine levels were also measured. In addition, we measured 8-hydroxy-2′-deoxyguanosine that is commonly used as a biomarker. This study shows, for the first time, that R-cdA and S-cdA exist in human urine and can be identified and quantified by LC-MS/MS. We propose that R-cdA and S-cdA may be well-suited biomarkers for disease processes such as carcinogenesis.