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1.
RNA ; 18(9): 1656-65, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22847815

ABSTRACT

The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.


Subject(s)
Methyltransferases/metabolism , RNA, Small Nucleolar/metabolism , Trichomonas vaginalis/enzymology , Base Sequence , Biological Transport , Guanosine/analogs & derivatives , Guanosine/metabolism , Methylation , Molecular Sequence Data , Nucleic Acid Conformation , RNA Caps/metabolism , RNA, Small Nucleolar/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment , Substrate Specificity , Trichomonas vaginalis/genetics
2.
Cell ; 142(2): 230-42, 2010 Jul 23.
Article in English | MEDLINE | ID: mdl-20655466

ABSTRACT

Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. Here, we report that TRF2 regulates the 5' exonuclease activity of its binding partner, Apollo, a member of the metallo-beta-lactamase family that is required for telomere integrity during S phase. TRF2 and Apollo also suppress damage to engineered interstitial telomere repeat tracts that were inserted far away from chromosome ends. Genetic data indicate that DNA topoisomerase 2alpha acts in the same pathway of telomere protection as TRF2 and Apollo. Moreover, TRF2, which binds preferentially to positively supercoiled DNA substrates, together with Apollo, negatively regulates the amount of TOP1, TOP2alpha, and TOP2beta at telomeres. Our data are consistent with a model in which TRF2 and Apollo relieve topological stress during telomere replication. Our work also suggests that cellular senescence may be caused by topological problems that occur during the replication of the inner portion of telomeres.


Subject(s)
Antigens, Neoplasm/metabolism , DNA Repair Enzymes/metabolism , DNA Replication , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Telomere/metabolism , Telomeric Repeat Binding Protein 2/metabolism , Cellular Senescence , DNA Damage , Exodeoxyribonucleases , Humans , Protein Structure, Tertiary
3.
RNA ; 16(1): 211-20, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19926722

ABSTRACT

The Tgs proteins are structurally homologous AdoMet-dependent eukaryal enzymes that methylate the N2 atom of 7-methyl guanosine nucleotides. They have an imputed role in the synthesis of the 2,2,7-trimethylguanosine (TMG) RNA cap. Here we exploit a collection of cap-like substrates to probe the repertoire of three exemplary Tgs enzymes, from mammalian, protozoan, and viral sources, respectively. We find that human Tgs (hTgs1) is a bona fide TMG synthase adept at two separable transmethylation steps: (1) conversion of m(7)G to m(2,7)G, and (2) conversion of m(2,7)G to m(2,2,7)G. hTgs1 is unable to methylate G or m(2)G, signifying that both steps require an m(7)G cap. hTgs1 utilizes a broad range of m(7)G nucleotides, including mono-, di-, tri-, and tetraphosphate derivatives as well as cap dinucleotides with triphosphate or tetraphosphate bridges. In contrast, Giardia lamblia Tgs (GlaTgs2) exemplifies a different clade of guanine-N2 methyltransferase that synthesizes only a dimethylguanosine (DMG) cap structure and cannot per se convert DMG to TMG under any conditions tested. Methylation of benzyl(7)G and ethyl(7)G nucleotides by hTgs1 and GlaTgs2 underscored the importance of guanine N7 alkylation in providing a key pi-cation interaction in the methyl acceptor site. Mimivirus Tgs (MimiTgs) shares with the Giardia homolog the ability to catalyze only a single round of methyl addition at guanine-N2, but is distinguished by its capacity for guanine-N2 methylation in the absence of prior N7 methylation. The relaxed cap specificity of MimiTgs is revealed at alkaline pH. Our findings highlight both stark and subtle differences in acceptor specificity and reaction outcomes among Tgs family members.


Subject(s)
Methyltransferases/classification , Methyltransferases/metabolism , RNA Cap Analogs/metabolism , RNA Cap Analogs/pharmacology , RNA Caps/metabolism , Catalysis , Catalytic Domain/physiology , Giardia lamblia/enzymology , Giardia lamblia/metabolism , Guanosine/analogs & derivatives , Guanosine/metabolism , Humans , Hydrogen-Ion Concentration , Methylation , Methyltransferases/physiology , Mimiviridae/enzymology , Mimiviridae/metabolism , RNA Caps/classification , Substrate Specificity
4.
RNA ; 15(4): 666-74, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19218551

ABSTRACT

A 2,2,7-trimethylguanosine (TMG) cap is a signature feature of eukaryal snRNAs, telomerase RNAs, and trans-spliced nematode mRNAs. TMG and 2,7-dimethylguanosine (DMG) caps are also present on mRNAs of two species of alphaviruses (positive strand RNA viruses of the Togaviridae family). It is presently not known how viral mRNAs might acquire a hypermethylated cap. Mimivirus, a giant DNA virus that infects amoeba, encodes many putative enzymes and proteins implicated in RNA transactions, including the synthesis and capping of viral mRNAs and the promotion of cap-dependent translation. Here we report the identification, purification, and characterization of a mimivirus cap-specific guanine-N2 methyltransferase (MimiTgs), a monomeric enzyme that catalyzes a single round of methyl transfer from AdoMet to an m(7)G cap substrate to form a DMG cap product. MimiTgs, is apparently unable to convert a DMG cap to a TMG cap, and is thereby distinguished from the structurally homologous yeast and human Tgs1 enzymes. Nonetheless, we show genetically that MimiTgs is a true ortholog of Saccharomyces cerevisiae Tgs1. Our results hint that DMG caps can satisfy many of the functions of TMG caps in vivo. We speculate that DMG capping of mimivirus mRNAs might favor viral protein synthesis in the infected host.


Subject(s)
DNA Viruses/enzymology , Methyltransferases/chemistry , Adenosine/analogs & derivatives , Adenosine/pharmacology , Amino Acid Sequence , Animals , Enzyme Inhibitors/pharmacology , Guanosine/analogs & derivatives , Guanosine/metabolism , Humans , Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , Molecular Sequence Data , RNA Caps/metabolism , RNA, Messenger/metabolism , S-Adenosylhomocysteine/pharmacology , Sequence Alignment
5.
Structure ; 16(4): 501-12, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18400173

ABSTRACT

The RNA triphosphatase (RTPase) components of the mRNA capping apparatus are a bellwether of eukaryal taxonomy. Fungal and protozoal RTPases belong to the triphosphate tunnel metalloenzyme (TTM) family, exemplified by yeast Cet1. Several large DNA viruses encode metal-dependent RTPases unrelated to the cysteinyl-phosphatase RTPases of their metazoan host organisms. The origins of DNA virus RTPases are unclear because they are structurally uncharacterized. Mimivirus, a giant virus of amoeba, resembles poxviruses in having a trifunctional capping enzyme composed of a metal-dependent RTPase module fused to guanylyltransferase (GTase) and guanine-N7 methyltransferase domains. The crystal structure of mimivirus RTPase reveals a minimized tunnel fold and an active site strikingly similar to that of Cet1. Unlike homodimeric fungal RTPases, mimivirus RTPase is a monomer. The mimivirus TTM-type RTPase-GTase fusion resembles the capping enzymes of amoebae, providing evidence that the ancestral large DNA virus acquired its capping enzyme from a unicellular host.


Subject(s)
Acid Anhydride Hydrolases/chemistry , DNA Viruses/enzymology , Models, Molecular , Nucleotidyltransferases/chemistry , Viral Proteins/chemistry , Acid Anhydride Hydrolases/genetics , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Methyltransferases/chemistry , Molecular Sequence Data , Mutation , Nucleotidyltransferases/genetics , Protein Structure, Tertiary , Sequence Alignment , Viral Proteins/genetics
6.
J Mol Biol ; 372(3): 723-36, 2007 Sep 21.
Article in English | MEDLINE | ID: mdl-17686489

ABSTRACT

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase(DV), is involved in two of four steps required for the formation of the viral mRNA cap (7Me)GpppA(2'OMe), the guanine-N7 and the adenosine-2'O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2'O-methyltransferase (MTase) activity has been shown on capped (7Me+/-)GpppAC(n) RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 A to 2.8 A resolution of NS5MTase(DV) in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of (7Me)GpppAC(4) and (7Me)GpppAC(5) RNAs is inhibited in the presence of GTP, (7Me)GTP and (7Me)GpppA but not by ATP. This particular position of the cap is in accordance with the 2'O-methylation step. A model was generated of a ternary 2'O-methylation complex of NS5MTase(DV), (7Me)GpppA and AdoMet. RNA-binding increased when (7Me+/-)GpppAGC(n-1) starting with the consensus sequence GpppAG, was used instead of (7Me+/-)GpppAC(n). In the NS5MTase(DV)-GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5' end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2'O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.


Subject(s)
Dengue Virus/chemistry , Methyltransferases/metabolism , RNA Caps/chemistry , RNA Caps/metabolism , RNA/chemistry , Viral Proteins/chemistry , Viral Proteins/metabolism , Base Sequence , Binding Sites , Guanosine , Guanosine Triphosphate , Ligands , Methylation , Models, Biological , Models, Molecular , Protein Structure, Tertiary , RNA Cap Analogs , Structure-Activity Relationship
7.
Virology ; 353(1): 133-43, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16844179

ABSTRACT

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes a cornucopia of proteins with imputed functions in DNA replication, modification, and repair. Here we produced, purified, and characterized mimivirus DNA ligase (MimiLIG), an NAD+-dependent nick joining enzyme homologous to bacterial LigA and entomopoxvirus DNA ligase. MimiLIG is a 636-aa polypeptide composed of an N-terminal NAD+ specificity module (domain Ia), linked to nucleotidyltransferase, OB-fold, helix-hairpin-helix, and BRCT domains, but it lacks the tetracysteine Zn-binding module found in all bacterial LigA enzymes. MimiLIG requires conserved domain Ia residues Tyr36, Asp46, Tyr49, and Asp50 for its initial reaction with NAD+ to form the ligase-AMP intermediate, but not for the third step of phosphodiester formation at a preadenylylated nick. MimiLIG differs from bacterial LigA enzymes in that its activity is strongly dependent on the C-terminal BRCT domain, deletion of which reduced its specific activity in nick joining by 75-fold without affecting the ligase adenylylation step. The DeltaBRCT mutant of MimiLIG was impaired in sealing at a preadenylylated nick. We propose that eukaryal DNA viruses acquired the NAD+-dependent ligases by horizontal transfer from a bacterium and that MimiLIG predates entomopoxvirus ligase, which lacks both the tetracysteine and BRCT domains. We speculate that the dissemination of NAD+-dependent ligase from bacterium to eukaryotic virus might have occurred within an amoebal host.


Subject(s)
Acanthamoeba/virology , DNA Ligases/chemistry , DNA Ligases/metabolism , Entomopoxvirinae/chemistry , Entomopoxvirinae/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Conserved Sequence , Cysteine/chemistry , Cysteine/metabolism , DNA Ligases/genetics , DNA Ligases/isolation & purification , Entomopoxvirinae/genetics , Molecular Sequence Data , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
8.
Virology ; 351(1): 145-58, 2006 Jul 20.
Article in English | MEDLINE | ID: mdl-16631221

ABSTRACT

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product formation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation.


Subject(s)
Flaviviridae/enzymology , RNA, Viral/biosynthesis , RNA-Dependent RNA Polymerase/metabolism , Amino Acid Sequence , Dengue Virus/enzymology , Gene Expression Regulation, Viral , Molecular Sequence Data , RNA-Dependent RNA Polymerase/chemistry , RNA-Dependent RNA Polymerase/genetics , West Nile virus/enzymology
9.
J Virol ; 80(1): 314-21, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16352556

ABSTRACT

Mimivirus, a parasite of Acanthamoeba polyphaga, is the largest DNA virus known; it encodes dozens of proteins with imputed functions in nucleic acid transactions. Here we produced, purified, and characterized mimivirus DNA topoisomerase IB (TopIB), which we find to be a structural and functional homolog of poxvirus TopIB and the poxvirus-like topoisomerases discovered recently in bacteria. Arginine, histidine, and tyrosine side chains responsible for TopIB transesterification are conserved and essential in mimivirus TopIB. Moreover, mimivirus TopIB is capable of incising duplex DNA at the 5'-CCCTT cleavage site recognized by all poxvirus topoisomerases. Based on the available data, mimivirus TopIB appears functionally more akin to poxvirus TopIB than bacterial TopIB, despite its greater primary structure similarity to the bacterial TopIB group. We speculate that the ancestral bacterial/viral TopIB was disseminated by horizontal gene transfer within amoebae, which are permissive hosts for either intracellular growth or persistence of many present-day bacterial species that have a type IB topoisomerase.


Subject(s)
DNA Topoisomerases, Type I/metabolism , Entomopoxvirinae/enzymology , Gene Transfer, Horizontal , Genome, Viral , Acanthamoeba/virology , Amino Acid Sequence , Animals , Bacteria/genetics , Bacteria/metabolism , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/isolation & purification , DNA, Superhelical/metabolism , Entomopoxvirinae/chemistry , Entomopoxvirinae/genetics , Molecular Sequence Data , Nucleic Acid Conformation
10.
Virology ; 328(2): 208-18, 2004 Oct 25.
Article in English | MEDLINE | ID: mdl-15464841

ABSTRACT

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.


Subject(s)
Acid Anhydride Hydrolases/metabolism , Dengue Virus/enzymology , Magnesium/metabolism , Nucleoside-Triphosphatase/metabolism , RNA Helicases/metabolism , Viral Nonstructural Proteins/metabolism , Virus Replication , Amino Acid Motifs , Amino Acid Sequence , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Helicases/chemistry , Sequence Alignment , Viral Nonstructural Proteins/genetics
11.
J Biol Chem ; 279(34): 35638-43, 2004 Aug 20.
Article in English | MEDLINE | ID: mdl-15152003

ABSTRACT

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase(DV)). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase(DV). A structural view of the binding of ribavirin 5'-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTase(DV), ribavirin 5'-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 A. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5'-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.


Subject(s)
Methyltransferases/chemistry , Nucleotides/chemistry , Binding Sites , Dengue/drug therapy , Dengue Virus/enzymology , Methyltransferases/antagonists & inhibitors , Models, Molecular , Nucleotides/pharmacology , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Viral Proteins/antagonists & inhibitors , Viral Proteins/chemistry
12.
EMBO J ; 21(11): 2757-68, 2002 Jun 03.
Article in English | MEDLINE | ID: mdl-12032088

ABSTRACT

Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 A resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2'-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses.


Subject(s)
Methyltransferases/metabolism , RNA/metabolism , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/physiology , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Dose-Response Relationship, Drug , Guanosine Triphosphate/metabolism , Models, Molecular , Molecular Sequence Data , Nucleotides/chemistry , Protein Binding , Protein Folding , Protein Structure, Tertiary , Time Factors
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