Properties of adenovirus messenger ribonucleic Acid synthesized in vitro

Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 μg of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 μg of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).     

Characterization of deoxyribonucleic Acid species from castor bean endosperm: inability to detect a unique deoxyribonucleic Acid species associated with glyoxysomes

Three DNA buoyant density species (nuclear, 1.692 g cm⁻³; mitochondria 1.705 g cm⁻³; and proplastid, 1.713 g cm⁻³) can be detected in extracts from castor bean endosperm. No other buoyant density species can be identified. DNA extracts from sucrose density gradient purified glyoxysomes exhibit varying amounts of each of the three identified DNAs but no other distinguishable DNA species. RNA synthesized in vitro by Escherichia coli RNA polymerase using purified castor bean nuclear DNA as a template, hybridizes equally well with its template and with the 1.692 g cm⁻³ species from glyoxysome fractions. These results are discussed in terms of their relevance to microbody biogenesis.     

Common Sequence at the 5′ Ends of the Segmented RNA Genomes of Influenza A and B Viruses

Guanylyl- and methyltransferases, isolated from purified vaccinia virus, were used to specifically label the 5′ ends of the genome RNAs of influenza A and B viruses. All eight segments were labeled with [α-³²P]guanosine 5′-triphosphate or S-adenosyl[methyl-³H]methionine to form “cap” structures of the type m⁷G(5′)pppN^m-, of which unmethylated (p)ppN- represents the original 5′ end. Further analyses indicated that m⁷G(5′)pppA^m, m⁷G(5′)pppA^mpGp, and m⁷G(5′)pppA^mpGpUp were released from total and individual labeled RNA segments by digestion with nuclease P1, RNase T1, and RNase A, respectively. Consequently, the 5′-terminal sequences of most or all individual genome RNAs of influenza A and B viruses were deduced to be (p)ppApGpUp. The presence of identical sequences at the ends of RNA segments of both types of influenza viruses indicates that they have been specifically conserved during evolution.     

Transformation of 2,2′-Bimorphine to the Novel Compounds 10-α-S-Monohydroxy-2,2′-Bimorphine and 10,10′-α,α′-S,S′-Dihydroxy-2,2′-Bimorphine by Cylindrocarpon didymum

Whole-cell suspensions of Cylindrocarpon didymum were observed to transform 2,2′-bimorphine to the compounds 10-α-S-monohydroxy-2,2′-bimorphine and 10,10′-α,α′-S,S′-dihydroxy-2,2′-bimorphine. Mass spectrometry and ¹H nuclear magnetic resonance spectroscopy confirmed the identities of these new morphine alkaloids.     

Reovirus Replicase-Directed Synthesis of Double-Stranded Ribonucleic Acid

After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand (³H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of ³H-cytidine triphosphate (or ³H-uridine triphosphate) and adenine triphosphate-α-³²P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3′-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of ³H to ³²P in these fragments indicated that RNA synthesis proceeded from the 5′ to 3′ terminus.     

Blocked and unblocked 5' termini in reovirus genome RNA

Uniformly ³²P-labeled, double-stranded genome RNA isolated from purified reovirus contains two types of 5′-terminal sequences. One strand contains a phosphatase-resistant 5′-terminal structure, XpppG^*pCpU, which is also present in the viral mRNA. The 5′ blocking group, X, is removed by β-elimination indicating that it is a nucleoside containing free 2′,3′-hydroxyls. G^*pC is an alkaline-resistant, 2′-O-methylated sequence. The other strand contains a phosphatase-sensitive 5′ sequence, ppGpPupPyp. The results are discussed in relation to blocked 5′-terminal structures in other viral and cellular RNAs.     

Effects of DNA3′pp5′G capping on 3′ end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance

When DNA breakage results in a 3′-PO₄ terminus, the end is considered ‘dirty’ because it cannot prime repair synthesis by DNA polymerases or sealing by classic DNA ligases. The noncanonical ligase RtcB can guanylylate the DNA 3′-PO₄ to form a DNA_3′pp_5′G_OH cap. Here we show that DNA capping precludes end joining by classic ATP-dependent and NAD⁺-dependent DNA ligases, prevents template-independent nucleotide addition by mammalian terminal transferase, blocks exonucleolytic proofreading by Escherichia coli DNA polymerase II and inhibits proofreading by E. coli DNA polymerase III, while permitting templated DNA synthesis from the cap guanosine 3′-OH primer by E. coli DNA polymerase II (B family) and E. coli DNA polymerase III (C family). Human DNA polymerase β (X family) extends the cap primer predominantly by a single templated addition step. Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA. We find that the embedded ppG is refractory to surveillance and incision by RNase H2.     

RecJ-like protein from Pyrococcus furiosus has 3′–5′ exonuclease activity on RNA: implications for proofreading of 3′-mismatched RNA primers in DNA replication

Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3′-mismatched RNA primer because it cannot remove the 3′-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3′-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3′–5′ direction, and the kinetic parameters (K_m and K_cat) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3′-mismatched RNA primers. Replication protein A, the single-stranded DNA–binding protein, stimulates the removal of 3′-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3′-mismatched RNA primer after the 3′-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3′-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo.     

Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [α-³²P]GDP in the presence of T1 ribonuclease the 3′-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA^Met_f; the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [α-³²P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.     

Isolation and properties of highly purified Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polumerase

1. The subunits α and β of Halobacterium cutirubrum DNA-dependent RNA polymerase have been purified to electrophoretic homogeneity. Both have mol.wt. 18000 and they are required in equimolar amounts for optimum activity. 2. The instability of the complete enzyme, αβ, in the absence of salt is due to the rapid inactivation of the β subunit in these conditions. 3. Nearest-neighbour analysis of the product formed on poly[d(A-T)] as template shows that the enzyme copies the latter accurately. 4. The enzyme initiates new chains with purine nucleoside triphosphates exclusively. 5. The product obtained in the standard assay conditions contains some high mol.wt. (>16S) material, but consists primarily of short chains, of average length 70–80 nucleotide units. 6. The template specificity of the complete enzyme has been studied at high and low ionic strength. Its extreme dependence on salt concentration is unrelated to the gross overall base composition of the DNA used. 7. T₇ DNA is transcribed asymmetrically and the enzyme selectively copies the T₇ `early' genes. 8. Preliminary amino acid analyses of α and β subunits show that their overall content of acidic, basic and neutral amino acids does not differ appreciably from that of Escherichia coli RNA polymerase.     

Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion

REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N ²-dG, (–)-trans-anti-benzo[a]pyrene-N ²-dG and 1,N ⁶-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N ² -dG adducts and the 1,N ⁶-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass.     

Structures of Bacterial Polynucleotide Kinase in a Michaelis Complex with Nucleoside Triphosphate (NTP)-Mg2+ and 5′-OH RNA and a Mixed Substrate-Product Complex with NTP-Mg2+ and a 5′-Phosphorylated Oligonucleotide

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5′-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5′-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg²⁺ and a 5′-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2′-hydroxyls of the 5′ terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5′-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg²⁺ and a 5′-OH DNA yielded a mixed substrate-product complex with GTP-Mg²⁺ and 5′-PO₄ DNA, wherein the product 5′ phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed.     

Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe

As predicted by the amino acid sequence, the purified protein coded by Schizosaccharomyces pombe SPAC2F7.06c is a DNA polymerase (SpPol4) whose biochemical properties resemble those of other X family (PolX) members. Thus, this new PolX is template-dependent, polymerizes in a distributive manner, lacks a detectable 3′→5′ proofreading activity and its preferred substrates are small gaps with a 5′-phosphate group. Similarly to Polμ, SpPol4 can incorporate a ribonucleotide (rNTP) into a primer DNA. However, it is not responsible for the 1–2 rNTPs proposed to be present at the mating-type locus and those necessary for mating-type switching. Unlike Polμ, SpPol4 lacks terminal deoxynucleotidyltransferase activity and realigns the primer terminus to alternative template bases only under certain sequence contexts and, therefore, it is less error-prone than Polμ. Nonetheless, the biochemical properties of this gap-filling DNA polymerase are suitable for a possible role of SpPol4 in non-homologous end-joining. Unexpectedly based on sequence analysis, SpPol4 has deoxyribose phosphate lyase activity like Polβ and Polλ, and unlike Polμ, suggesting also a role of this enzyme in base excision repair. Therefore, SpPol4 is a unique enzyme whose enzymatic properties are hybrid of those described for mammalian Polβ, Polλ and Polμ.     

Error-prone lesion bypass by human DNA polymerase eta

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N²-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N²-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans.     

Two novel dATP analogs for DNA photoaffinity labeling

Two new photoreactive dATP analogs, N⁶-[4-azidobenzoyl–(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (AB-dATP) and N⁶-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl)]-2′-deoxyadenosine-5′-triphosphate (DB-dATP), were synthesized from 2′-deoxyadenosine-5′-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N¹-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate to N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-monophosphate (N⁶-dAMP). Next, N⁶-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N⁶-(2-aminoethyl)-2′-deoxyadenosine-5′-triphosphate (N⁶-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N⁶-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.     

Identification of delta helicase as the bovine homolog of HUPF1: demonstration of an interaction with the third subunit of DNA polymerase delta

Delta helicase is a 5′ to 3′ DNA helicase that partially co-purifies with DNA polymerase delta (pol delta) from fetal bovine thymus tissue. We describe the resolution of delta helicase from pol delta on heparin–agarose chromatography and its purification to apparent homogeneity by affinity purification on single-stranded DNA–cellulose chromatography, unique-sequence RNA–agarose chromatography, and ceramic hydroxyapatite chromatography. Delta helicase isolated from fetal bovine thymus had an apparent M_r of 115 kDa in SDS–PAGE, and photo-crosslinked to [α-³²P]ATP. Tandem mass spectrometry peptide mass data derived from the bovine polypeptide matched to human UPF1 (HUPF1), a 5′ to 3′ RNA and DNA helicase, and a requisite component of the mRNA surveillance complex. Antisera against HUPF1 cross-reacted with delta helicase on western analysis, and delta helicase activity was immunoinactivated by pre-incubation with antibodies to HUPF1, suggesting that delta helicase is the bovine homolog of HUPF1. Immunoprecipitation experiments demonstrated that HUPF1 interacts with the 66-kDa third subunit of pol delta in vivo.