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PubMed2004

Stable down-regulation of human polynucleotide kinase enhances spontaneous mutation frequency and sensitizes cells to genotoxic agents.

Human polynucleotide kinase (hPNK) is a 57.1-kDa monomeric protein with conserved motifs associated with phosphatase and kinase activities. hPNK catalyzes phosphorylation of 5'-DNA termini and dephosphorylation of 3'-DNA termini. Previous studies, employing cell-free systems, have suggested that hPNK participates in the repair of DNA strand breaks. To better define the cellular function of hPNK, a double-stranded small-interfering RNA molecule designed to stably target hPNK transcription was introduced into A549 human lung adenocarcinoma cells. The small-interfering RNA suppressed hPNK gene expression by at least 80-90%. These cells exhibited a 7-fold higher spontaneous mutation frequency based on the development of resistance to ouabain; elevated sensitivity to a broad range of genotoxic agents including gamma-radiation, UVC radiation, methyl methanesulfonate, hydrogen peroxide, and camptothecin; and slower repair of radiation-induced DNA strand breaks. These findings underscore the importance of hPNK in the maintenance of DNA integrity after damage induced by endogenous and exogenous agents.

DNA Damage↗
PubMed1970

Polynucleotide ligase-catalyzed joining of deoxyribo-oligonucleotides on ribopolynucleotide templates and of ribo-oligonucleotides on deoxyribopolynucleotide templates.

T4 polynucleotide ligase efficiently catalyzes the head-to-tail joining of the ribo-oligoadenylates, r-(pA)(8) and r-(pA)(10), in the presence of high molecular weight deoxypolythymidylate. The enzyme also catalyzes the joining of deoxy-oligothymidylates, e.g., d-(pT)(10), in the presence of ribopolyadenylate. The enzyme failed to bring about the joining of r-(pA)(10) when poly r-U was used as the template, although a slow formation of the expected activated intermediate from r-(pA)(10) was detected.

Adenine Nucleotides↗
PubMed1981

tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains.

Adenosine Triphosphate↗
PubMed1997

Functional overlap of tRNA nucleotidyltransferase, poly(A) polymerase I, and polynucleotide phosphorylase.

Repair of the 3'-terminal -CCA sequence of tRNA generally requires the action of the enzyme tRNA nucleotidyltransferase. However, in Escherichia coli in the absence of this enzyme, a decreased level of tRNA end repair continues. To ascertain the enzymes responsible for this residual repair, mutant strains were constructed lacking tRNA nucleotidyltransferase and other enzymes potentially involved in the process, poly(A) polymerase I and polynucleotide phosphorylase (PNPase). Strains lacking tRNA nucleotidyltransferase and either one of the other enzymes displayed decreased growth rates and increased levels of defective tRNA compared with the single cca mutant. Triple mutants lacking all three enzymes grew very slowly, had even more defective tRNA, and were devoid of activity incorporating AMP into tRNA-C-C. Overexpression of poly(A) polymerase I, but not PNPase, partially compensated for the absence of tRNA nucleotidyltransferase. These data show that poly(A) polymerase I and PNPase participate in the end repair process and are required to maintain functional tRNA levels when tRNA nucleotidyltransferase is absent.

Adenosine Monophosphate↗
PubMed1998

Polynucleotide phosphorylase is a component of a novel plant poly(A) polymerase.

We have isolated cDNA clones encoding a novel RNA-binding protein that is a component of a multisubunit poly(A) polymerase from pea seedlings. The encoded protein bears a significant resemblance to polynucleotide phosphorylases (PNPases) from bacteria and chloroplasts. More significantly, this RNA-binding protein is able to degrade RNAs with the resultant production of nucleotide diphosphates, and it can add extended polyadenylate tracts to RNAs using ADP as a donor for adenylate moieties. These activities are characteristic of PNPase. Antibodies raised against the cloned protein simultaneously immunoprecipitate both poly(A) polymerase and PNPase activity. We conclude from these studies that PNPase is the RNA-binding cofactor for this poly(A) polymerase and is an integral player in the reaction catalyzed by this enzyme. The identification of this RNA-binding protein as PNPase, which is a chloroplast-localized enzyme known to be involved in mRNA 3'-end determination and turnover (Hayes, R., Kudla, J., Schuster, G., Gabay, L., Maliga, P., and Gruissem, W. (1996) EMBO J. 15, 1132-1141), raises interesting questions regarding the subcellular location of the poly(A) polymerase under study. We have reexamined this issue, and we find that this enzyme can be detected in chloroplast extracts. The involvement of PNPase in polyadenylation in vitro provides a biochemical rationale for the link between chloroplast RNA polyadenylation and RNA turnover which has been noted by others (Lisitsky, I., Klaff, P., and Schuster, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13398-13403).

Amino Acid Sequence↗
PubMed2001

Domain structure and mutational analysis of T4 polynucleotide kinase.

T4 polynucleotide kinase (Pnk) is the founding member of a family of 5'-kinase/3'-phosphatase enzymes that heal broken termini in RNA or DNA by converting 3'-PO(4)/5'-OH ends into 3'-OH/5'-PO(4) ends, which are then suitable for sealing by RNA or DNA ligases. Here we employed site-directed mutagenesis and biochemical methods to dissect the domain structure of the homotetrameric T4 Pnk protein and to localize essential constituents of the apparently separate active sites for the 5'-kinase and 3'-phosphatase activities. We characterized deletion mutants Pnk(42-301) and Pnk(1-181), which correspond to domains defined by proteolysis with chymotrypsin. Pnk(1-181) is a monomer with no 3'-phosphatase and low residual 5'-kinase activity. Pnk(42-301) is a dimer with no 5'-kinase and low residual 3'-phosphatase activity. Four classes of missense mutational effects were observed. (i) Mutations K15A, S16A, and D35A inactivated the 5'-kinase but did not affect the 3'-phosphatase or the tetrameric quaternary structure of T4 Pnk. 5'-kinase activity was ablated by the conservative mutations K15R, K15Q, and D35N; however, kinase activity was restored by the S16T change. (ii) Mutation D167A inactivated the 3'-phosphatase without affecting the 5'-kinase or tetramerization. (iii) Mutation D85A caused a severe decrement in 5'-kinase activity and only a modest effect on the 3'-phosphatase; the nearby N87A mutation resulted in a significantly reduced 3'-phosphatase activity and slightly reduced 5'-kinase activity. D85A and N87A both affected the quaternary structure, resulting in a mixed population of tetramer and dimer species. (iv) Alanine mutations at 11 other conserved positions had no significant effect on either 5'-kinase or 3'-phosphatase activity.

Alanine↗
PubMed2004

Characterization of polynucleotide kinase/phosphatase enzymes from Mycobacteriophages omega and Cjw1 and vibriophage KVP40.

Coliphage T4 Pnkp is a bifunctional polynucleotide 5'-kinase/3'-phosphatase that catalyzes the end-healing steps of a RNA repair pathway. Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5'-kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3'-phosphatase module with an essential acyl-phosphatase motif, DX- DGT. Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/kinase enzymes found in mammals and fission yeast. Whereas the phage 5'-kinases differ from each other in their preferences for phosphorylation of 5' overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase. We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response. Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo. A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.

Adenosine Triphosphate↗
PubMed2005

Human mitochondrial mRNAs are stabilized with polyadenylation regulated by mitochondria-specific poly(A) polymerase and polynucleotide phosphorylase.

Mammalian mitochondrial (mt) mRNAs have short poly(A) tails at their 3' termini that are post-transcriptionally synthesized by mt poly(A) polymerase (PAP). The polyadenylation of mt mRNAs is known to be a key process needed to create UAA stop codons that are not encoded in mtDNA. In some cases, polyadenylation is required for the tRNA maturation by editing of its 3' terminus. However, little is known about the functional roles the poly(A) tail of mt mRNAs plays in mt translation and RNA turnover. Here we show human mt PAP (hmtPAP) and human polynucleotide phosphorylase (hPNPase) control poly(A) synthesis in human mitochondria. Partial inactivation of hmtPAP by RNA interference using small interfering RNA in HeLa cells resulted in shortened poly(A) tails and decreased steady state levels of some mt mRNAs as well as their translational products. Moreover, knocking down hmtPAP generated markedly defective mt membrane potentials and reduced oxygen consumption. In contrast, knocking down hPNPase showed significantly extended poly(A) tails of mt mRNAs. These results demonstrate that the poly(A) length of human mt mRNAs is controlled by polyadenylation by hmtPAP and deadenylation by hPNPase, and polyadenylation is required for the stability of mt mRNAs.

Base Sequence↗
PubMed2006

Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase.

The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp-392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu.

Binding Sites↗
PubMed1987

Interpretation of DNA vibration modes. II--The adenosine and thymidine residues involved in oligonucleotides and polynucleotides.

Normal coordinate analysis of the adenosine and thymidine residues involved in the right- and left-handed conformations of oligonucleotides and polynucleotides has been performed. The valence force field, employed in this work, allowed recently to reproduce the vibrational spectra of 2'-deoxythymidine and 2'-deoxyadenosine. The calculated wavenumbers based on a non-redundant set of internal coordinates have been compared to the Raman and infrared peak positions arising from A, B, C, D and Z conformations, in the 1550-1250 cm-1 and 800-600 cm-1 spectral regions: i.e. characteristic of adenosine and thymidine residues. Moreover, a systematic study has been performed on the evolution of the vibrational wavenumbers as a function of the glycosidic angle (chi) and the sugar pucker conformation.

Adenosine↗
PubMed1991

Triple helical polynucleotidic structures: sugar conformations determined by FTIR spectroscopy.

Fourier Transform Infrared Spectra of triple stranded polynucleotides containing homopurine dA or rA and homopyrimidine dT or rU strands have been obtained in H2O and D2O solutions as well as in hydrated films at various relative humidities. The spectra are interpreted by comparison with those of double stranded helixes with identical base and sugar composition. The study of the spectral domain corresponding to in-plane double bond stretching vibrations of the bases shows that whatever the initial duplex characterized by a different IR spectrum (A family form poly rA.poly rU, heternomous form poly rA.poly dT, B family form poly dA.poly dT), the triplexes present a similar IR spectrum reflecting similar base interactions. A particular attention is devoted to the 950-800 cm-1 region which contains marker bands of the sugar conformation in the nucleic acids. In solution the existence of only N (C3'endo-A family form) type of sugar pucker is detected in poly rU.poly rA.poly rU and poly dt.poly rA.poly rU. On the contrary absorption bands characteristic of both N (C3'endo-A family form) and S (C2'endo-B family form) type sugars are detected for poly rU.poly rA.poly dT, poly rU.poly dA.poly dT and poly dT.poly rA.poly dT. Finally mainly S (C2'endo-B family form) type sugars are observed in poly dT.poly dA.poly dT.

Carbohydrates↗
PubMed1995

Hypericin site specific interactions within polynucleotides used as DNA model compounds.

Interactions of the antiretroviral hypericin molecule with polynucleotides, i.e. poly(dG-dC), poly(dA-dT), poly(rG) and poly(rC), have been studied in aqueous solutions by resonance Raman spectroscopy, using an UV excitation wavelength which induces a specific resonance enhancement of spectral band intensities corresponding to proper nucleic base modes of vibration. It is shown that: i) hypericin selectively interacts with the N7 sites of purines, ii) the strength of interaction depends on the polymer structure, and: iii) interaction with guanine is stronger than with adenine molecules.

Anthracenes↗
PubMed1996

Tryptophan intercalation in G, C containing polynucleotides: Z to B conversion of poly [d(G-5M C)] in low salt induced by a tetra peptide.

Binding of a tetra peptide, lysyl tryptophenyl glycyl lysine O-ter butyl ester (KWGK) with duplex forms of G, C containing polynucleotides, Poly [d(G-C)], Poly [d(G-5M C)], Poly (dG), Poly (dC) and E.coli DNA were studied under low salt conditions using UV absorption, fluorescence, and circular dichroic (C.D) spectroscopy. On addition of the peptide (upto a P/N mole ratio of 0.5), the Poly [d(G-5M C)] under low salt (1 mM Na Cl) conditions, was converted from Z to B-form as shown by the inversion of C.D spectra. The two binding constants (K1 and K2) were determined from fluorescence spectroscopy of which K2 estimates the intercalation of the tryptophenyl side chain between the base pairs of DNA and K1 estimates the electrostatic interactions between the lysyl side chains and phosphate groups. The strength of intercalation is: Z-form of Poly [d(G-5M C)] >> B form of Poly [d(G-5M C)] >> E.Coli DNA > Poly (dG).Poly (dC). This means that peptide seems to have strong preference for Z compared to B-form and for alternating over non-alternating G, C Sequences. This suggests that tryptophan intercalation may act as a discriminating factor in recognizing Z and B-forms and may have a potential role in Protein-Nucleic acid interactions that are important for transcription.

Binding Sites↗
PubMed1971

Regulation of the immune system by synthetic polynucleotides. II. Action on peritoneal exudate cells.

Incubation of antigen with normal mouse peritoneal exudate cells in vitro and subsequent reinjection of the washed cells into syngeneic mice resulted in increased antibody titers as compared to mice injected with antigen alone. Several of the variables influencing this system were studied with and without the stimulus of complex homoribopolynucleotides (poly A:U or poly I:C) as adjuvants to determine the cellular site of action of the latter. It was found that addition of poly A:U or poly I:C caused a further rise in circulating antibody levels which correlated with increased RNA synthesis, suggesting that the macrophage was one cell affected by this adjuvant. Actinomycin D was found to inhibit the rise in titer induced by PEC and this inhibition could be overcome by poly A:U. Injection of the polynucleotides 18 hr before antigen resulted in depression of circulating antibody levels, and poly A:U or poly I:C injected 18 hr before harvesting PEC and incubation with antigen also inhibited the capacity of the PEC to increase antibody levels. A 4S RNA-rich fraction was purified after treatment with phenol of PEC exposed to antigen in vitro, and under the stimulus of poly A:U this RNA was capable of inducing specific antibody titers and rosette-forming cells on injection into mice. Antigen contamination of Pronase-treated RNA, active biologically, was below 10(-11) g as determined isotopically.

Adenine Nucleotides↗
PubMed2004

Translocation of structured polynucleotides through nanopores.

We investigate theoretically the translocation of structured RNA/DNA molecules through narrow pores which allow single but not double strands to pass. The unzipping of basepaired regions within the molecules presents significant kinetic barriers for the translocation process. We show that this circumstance may be exploited to determine the full basepairing pattern of polynucleotides, including RNA pseudoknots. The crucial requirement is that the translocation dynamics (i.e. the length of the translocated molecular segment) needs to be recorded as a function of time with a spatial resolution of a few nucleotides. This could be achieved, for instance, by applying a mechanical driving force for translocation and recording force-extension curves (FECs) with a device such as an atomic force microscope or optical tweezers. Our analysis suggests that, with this added spatial resolution, nanopores could be transformed into a powerful experimental tool to study the folding of nucleic acids.

Animals↗
PubMed1994

Inhibition of T4 polynucleotide kinase activity by phosphorothioate and chimeric oligodeoxynucleotides.

Whole and partially modified phosphorothioate oligodeoxynucleotides (ODN) were found to directly inhibit T4 polynucleotide kinase (PNK) activity, while phosphodiester ODN showed no detectable inhibition. This inhibition was found to be length dependent, as demonstrated by a 28-mer phosphorothioate ODN with an IC50 of 12 nM, and an 8-mer phosphorothioate ODN with an IC50 of 27,000 nM. Inhibition depended on the number and type of modified internucleotide linkages: a 20-mer phosphorothioate ODN had an IC50 of 21 nM, while a chimeric ODN with seven phosphorothioate linkages and an identical sequence showed no inhibition. On the other hand, the same sequence as a chimeric phosphorodithioate ODN (with seven dithioate linkages) had an IC50 of 580 nM. Four different chimeric phosphorodithioate ODN showed markedly different potencies of inhibition, suggesting that inhibition of PNK activity can be sequence specific.

Bacteriophage T4↗
PubMed1989

Binding of 7H-dibenzo[c,g]carbazole to polynucleotides and DNA in vitro.

The N-heterocyclic aromatic pollutant, 7H-dibenzo[c,g]carbazole (DBC), is a potent carcinogen having both local and systemic effects. The overall objective of this research was to investigate the nature of the covalent binding of DBC with nucleic acids in vitro. DBC was shown to bind to polynucleotides, RNA and DNA in an in vitro rat or hamster microsomal enzyme assay, exhibiting a preferential binding to polyguanylic acid (poly[G]). Benzo[a]pyrene (BaP) binding to these same nucleic acids was determined simultaneously and was approximately 10-fold higher than DBC binding under identical experimental conditions. DBC-nucleic acid binding was shown to be dependent upon the presence of a microsomal activating system, the results being similar for rat or hamster liver microsomes. This microsome-dependent binding was unaffected by the addition of epoxide hydrase activity modifiers but was almost completely inhibited by alpha-naphthoflavone. The nature of DBC-nucleic acid binding was investigated using fluorescence spectroscopy. Benzo[c]carbazole and 5,5,6,6-tetrahydrodibenzo[c,g]carbazole were synthesized as representatives of the effect of disruption of the DBC pi-electron system on fluorescence excitation and emission. DBC-poly[G] adducts were isolated from binding assay mixtures and separated by HPLC. Results indicated that there are at least three different DBC-poly[G] adducts formed in vitro. The emission spectra of isolated adducts were similar in shape to that of DBC; however, the adduct spectra were shifted 5-10 nm toward longer wavelengths. This suggests that the bound DBC species have intact pi-electron systems. Results are consistent with binding through the nitrogen position as well as binding through the 1,2,3,4-ring of the molecule.

Animals↗
PubMed1991

Spectroscopic characteristics and site I/site II classification of cis and trans benzo[a]pyrene diolepoxide enantiomer-guanosine adducts in oligonucleotides and polynucleotides.

The highly tumorigenic isomer (+)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] and its non-tumorigenic enantiomer (-)-anti-BPDE are known to react predominantly with the exocyclic amino group (N2) of deoxyguanine in DNA and to form adducts of different conformations. The spectroscopic characteristics (UV absorbance, fluorescence and circular dichroism) of stereochemically defined (+)-trans, (-)-trans, (+)-cis and (-)-cis d(5'-CACATGBPDETACAC) adducts in the single-stranded form, or complexed with the complementary strand d(5'-GTGTACATGTG) in aqueous solution, were investigated. The spectroscopic characteristics of the double-stranded d(5'-CACATGBPDETACAC).d(5'-GTGTACATGTG) adducts can be interpreted in terms of two types of conformations. In site I-type conformations, there is an approximately 10 nm red shift in the absorption maxima, which is attributed to significant pyrenyl residue-base interactions; in site II-type adducts, the red shift is only approximately 2-3 nm, and the pyrene ring system is located at external, solvent-exposed binding sites. The spectroscopic characteristics of the BPDE-modified duplexes are of the site II type for the (+)- and (-)-trans, and of the site I type for the (+)- and (-)-cis adducts. In adducts derived from the binding of (+)-anti-BPDE to poly(dG-dC).(dG-dC) and poly(dG).(dC), the trans/cis BPDE-N2-dG adduct ratio is 6 +/- 1; in the case of (-)-anti-BPDE this ratio is only 0.4 +/- 0.1 and 0.6 +/- 0.15 in poly(dG-dC).(dG-dC) and poly(dG).(dC) respectively. The spectroscopic properties of these BPDE-modified polynucleotide adducts are consistent with those of the BPDE-modified oligonucleotide complexes; the cis adducts are correlated with site I adduct conformations, while the trans adducts are of the site II type. The correlations between adduct characteristics and biological activities of the two BPDE enantiomers are discussed.

Base Sequence↗