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At least 523 records · Page 29Linked to original sources
PubMed2002

Involvement of human polynucleotide kinase in double-strand break repair by non-homologous end joining.

The efficient repair of double-strand breaks (DSBs) in DNA is critical for the maintenance of genome stability. In mammalian cells, repair can occur by homologous recombination or by non-homologous end joining (NHEJ). DNA breaks caused by reactive oxygen or ionizing radiation often contain non- conventional end groups that must be processed to restore the ligatable 3'-OH and 5'-phosphate moieties which are necessary for efficient repair by NHEJ. Here, using cell-free extracts that efficiently catalyse NHEJ in vitro, we show that human polynucleotide kinase (PNK) promotes phosphate replacement at damaged termini, but only within the context of the NHEJ apparatus. Phosphorylation of terminal 5'-OH groups by PNK was blocked by depletion of the NHEJ factor XRCC4, or by an inactivating mutation in DNA-PK(cs), indicating that the DNA kinase activity in the extract is coupled with active NHEJ processes. Moreover, we find that end-joining activity can be restored to PNK-depleted extracts by addition of human PNK, but not bacteriophage T4 PNK. This work provides the first demonstration of a direct, specific role for human PNK in DSB repair.

Antigens, Nuclear↗
PubMed1982

Computational studies of polynucleotide flexibility.

Details of polynucleotide flexibility may be probed through a combination of semiempirical potential energy calculations and statistical mechanical analyses. The pseudorotational motions of the furanose and the long-range correlated rotations of the chain backbone are described briefly here.

Computers↗
PubMed1983

Possible conformations of double-helical polynucleotides containing incorrect base pairs.

Theoretical conformational analysis using classical potential functions has shown the possibility of incorporation of nucleotide mispairs with the bases in normal tautomeric forms into the DNA double helix. Incorrect purine-pyrimidine, purine-purine and pyrimidine-pyrimidine pairs can be incorporated into the double helix existing both in A- and B-conformations. The most energy favourable conformations of fragments containing a mispair have all the dihedral angles of the sugar-phosphate backbone within the limits characteristic of double helices consisting of Watson-Crick nucleotide pairs. Incorporation of mispairs is possible practically without the appearance of reduced interatomic contacts. Mutual position of bases in the incorporated mispair does not differ much from their position at the energy minimum of the corresponding isolated base pairs. Conformational parameters of irregular regions of double-stranded polynucleotides containing G:U, I:A, I:A* (syn) and U:C pairs are presented. Distortion of the sugar-phosphate backbone is the least upon incorporation of the G:U pair. Formation of mispairs in the processes of nucleic acid biosynthesis and spontaneous mutagenesis is discussed.

Base Composition↗
PubMed1986

Stabilization of T4 polynucleotide kinase by macromolecular crowding.

T4 polynucleotide kinase rapidly loses activity during its reaction on duplex DNA termini. Addition of high concentrations of nonspecific polymers reverses or prevents this inactivation. In contrast, additions of related materials of lower molecular weight are relatively ineffective in stabilizing the kinase. Such a pattern suggests that the stabilizing effects of polymers on kinase activity are due to macromolecular crowding. An effect of crowding on the known tendency of the kinase to undergo oligomerization reactions is consistent with our observations.

DNA↗
PubMed1975

Polynucleotide fragments from the 28S ribosomal RNA of insects.

If RNA is extracted from the ribosomes which had been isolated from frozen-thawed tissue of Galleria mellonella, the 28 S RNA, when heated or treated with urea, dissociates into seven different species of polynucleotide fragments. They were designated as R1, R2, R3, R4, R5, R6, and R7, whose molecular weights were estimated to be 1.15x10-6, 0.75x10-6, 0.55x10-6, 0.40x10-6, 0.30x10-6, 0.25x10-6, 0.20x10-6 daltons, respectively. It is likely that R1 and R5 arise from a single nick in original 38 S rRNA. Experiments with isolated R1 suggest that it is made up of a hydrogen-bonded complex of R2 and R4. R5 is a complex of R6 and an unidentified species, X. It is suggested that these fragments result from nicks which are introduced, secondarily, in the phosphodiester bonds by an endogenous endonuclease(s). Since the secondary nicks are limited in number and located in specific points of the molecule, it appears that the reaction is quite specific. It was also shown that the 28 S aphid RNA, which apparently lacks the primary nick, is susceptible to nicking.

Animals↗
PubMed1975

Hydrolysis in 2'-chloro-2'-deoxy nucleosides, -nucleotides and -polynucleotides to arabinose-derivatives.

The rate of hydrolysis of 2'-chloro-2'-deoxycytidine and -uridine, and their 5'-phosphates, -diphosphates and polymers to the corresponding arabinocytidine and arabinouridine derivatives in the presence of Tris-HCL pH 8.9 is determined. This rate is highest for the nucleosides, less for the 5'-phosphates and 5'-diphosphates and even less for the polynucleotides. In the the 2'-chloro-2'-deoxyuridine series the O2, 2'-cyclonucleoside was formed in addition to the arabinonucleoside.

Alkaline Phosphatase↗
PubMed1994

Sterical recognition by T4 polynucleotide kinase of non-nucleosidic moieties 5'-attached to oligonucleotides.

The ability of T4 polynucleotide kinase (PNK) to phosphorylate non-nucleosidic moieties 5'-attached to oligodeoxynucleotides (ODNs) has been investigated. Non-nucleosidic phosphoramidite units were prepared from ethane-1,2-diol and propane-1,3-diol backbones. Some of them corresponded to pure enantiomers. They were used to obtain the corresponding 5'-end modified oligothymidylates X(pdT)10. The free primary hydroxyl of the non-nucleosidic moieties (X) of these oligomers was phosphorylated by PNK. We report the stereoselective phosphorylation of the L form of the 5'-end attached non-nucleosidic chiral fragments; the non-chiral moieties were completely phosphorylated. Dimers of glycerol analogue and thymidine 3'-phosphate were not recognized by PNK and the shortest modified ODN able to be phosphorylated was a trinucleotide X(pdT)3. A modified X(pdT)10, bearing a cyclic abasic site (X) at its 5'-end, was prepared by chemical synthesis from 1,2-dideoxyribose phosphoramidite and was phosphorylated with a 90% yield.

Chromatography, High Pressure Liquid↗
PubMed2000

Hydration effects of Ni(2+) binding to synthetic polynucleotides with regularly alternating purine-pyrimidine sequences.

High precision ultrasonic and densimetric techniques have been used to study the interaction of Ni(2+)ions with right-handed poly[d(G-C)].poly[d(G-C)], poly-[d(A-C)].poly[d(G-T)] and poly[d(A-T)].poly[d(A-T)] in 5 mM CsCl, 0.2 mM HEPES, pH 7.5 at 20 degrees C. From these measurements the changes in the apparent molar volume and the apparent molar adiabatic compressibility due to the interaction have been obtained. The volume effects of the binding, calculated per mole of Ni(2+)ions, range from 11.7 to 23.9 cm(3)mol(-1)and the compressibility effects range from 19.3 x 10(-4)to 43.1 x 10(-4)cm(3)mol(-1)bar(-1). These data are interpreted in terms of dehydration of the polynucleotides and Ni(2+)ions, i.e. the release of water molecules from the hydration shells of the molecules. An increase in G+C content gives an increase in volume and compressibility effects, indicating a rise in the extent of dehydration. The dehydration effects of Ni(2+)binding to poly[d(G-C)].poly[d(G-C)] are approximately twice those of poly[d(A-T)].poly[d(A-T)]. The volume and compressibility effects of Ni(2+)-EDTA complex formation have also been measured and used as a model system for quantitative estimation. These values revealed that Ni(2+)ions can coordinate two atomic groups of poly[d(G-C)]. poly[d(G-C)], while in the case of the Ni(2+)-poly[d(A-T)]. poly[d(A-T)] complex volume and compressibility effects correspond to one direct or two indirect (through water) contacts.

Circular Dichroism↗
PubMed1976

Partial characterization of an endonuclease activity which appears in nuclease free T4 polynucleotide kinase.

A nuclease activity has been found to appear in preparations of T4 induced polynucleotide kinase which had originally been nuclease free. The nuclease introduced random nicks into T7 DNA suggesting that it was an endonuclease. Destabilization of the kinase molecule by osmotic shock or by the removal of reducing agents, ATP or salts was shown to stimulate the endonuclease appearance. The molecular weight was found to be 32,000 +/- 10% by gel filtration on G100 Sephadex. The nuclease was active over a wide pH range from pH 5.0 to pH 9.2 in a number of buffer systems and required MgCl2 and reducing agent for maximum activity. Sodium azide did not affect the nuclease appearance.

Coliphages↗
PubMed1976

Analysis of the possible helical structures of nucleic acids and polynucleotides. Application of (n-h) plots.

The two helical parameters n and h where n is the number of nucleotide residues per turn and h is the height per nucleotide residue have been evaluated for single stranded helical polynucleotide chains comprising C(3') -endo and C(2') endo class of nucleotides. The helical parameters are found to be especially sensitive to the C(4')-C(3') (sugar pucker) and the C(4')-C(5') torsions. The (n-h) plots display only one important helix forming domain for each class of nucleotides characterized by the sugar pucker and the C(4')-C(5') torsion. A correlation between the (n-h) plots and the known RNA (A,A') and DNA (A,B,C) helical forms has been established. It is found that all forms of helices except the C-DNA possess a favorable combination of P-O torsions. The analysis of the (n-h) plots suggests that C-DNA can have a conformation very similar to B-DNA. Although the (n-h) plots predict the stereochemical possibility of both right-handed and left-handed helices, nucleic acids apparently prefer right-handed conformation because of the energetics associated with the sugar-phosphate backbone and the base.

Binding Sites↗
PubMed2002

Mutational analysis defines the 5'-kinase and 3'-phosphatase active sites of T4 polynucleotide kinase.

T4 polynucleotide kinase (Pnk) is a bifunctional 5'-kinase/3'-phosphatase that aids in the repair of broken termini in RNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO4 ends, which are then sealed by RNA ligase. Here we have employed site-directed mutagenesis (introducing 31 mutations at 16 positions) to locate candidate catalytic residues within the 301 amino acid Pnk polypeptide. We found that alanine substitutions for Arg38 and Arg126 inactivated the 5'-kinase, but spared the 3'-phosphatase activity. Conservative substitutions of lysine or glutamine for Arg38 and Arg126 did not restore 5'-kinase activity. These results, together with previous mutational studies, highlight a constellation of five amino acids (Lys15, Ser16, Asp35, Arg38 and Arg126) that likely comprise the 5'-kinase active site. Four of these residues are conserved at the active sites of adenylate kinases (Adk), suggesting that Pnk and Adk are structurally and mechanistically related. We found that alanine substitutions for Asp165, Asp167, Arg176, Arg213, Asp254 and Asp278 inactivated the 3'-phosphatase, but spared the 5'-kinase. Conservative substitutions of asparagine or glutamate for Asp165, Asp167 and Asp254 did not revive the 3'-phosphatase activity, nor did lysine substitutions for Arg176 and Arg213. Glutamate in lieu of Asp278 partially restored activity, whereas asparagine had no salutary effect. Alanine substitutions for Arg246 and Arg279 partially inactivated the 3'-phosphatase; the conservative R246K change restored activity, whereas R279K had no benefit. The essential phosphatase residues Asp165 and Asp167 are located within a 165DxDxT169 motif that defines a superfamily of phosphotransferases. Our data suggest that the 3'-phosphatase active site incorporates multiple additional functional groups.

Alanine↗
PubMed1977

Phosphorylation of tRNA by T4 polynucleotide kinase.

The phosphorylation of various intact tRNA species by T4 polynucleotide kinase has been studied. The apparent Michaelis constant was on the average found to be 100 times lower than for some single-stranded DNAs previously studied. (J.R. Lillehaug and K. Kleppe, (1975) Biochemistry, 14, 1221). Conditions which result in complete phosphorylation of different tRNA species have also been established. Studies on equilibrium constants and the reversibility of the reaction revealed that the phosphorylation reaction is not a true equilibrium reaction under the conditions used in this work.

Coliphages↗
PubMed1978

Theoretical calculations of base-base interactions in nucleic acids: II. Stacking interactions in polynucleotides.

Base-base interactions were computed for single- and double stranded poly,ucleotides, for all possible base sequences. In each case, both right and left stacking arrangements are energetically possible. The preference of one over the other depends upon the base-sequence and the orientation of the bases with respect to helix-axis. Inverted stacking arrangement is also energetically possible for both single- and double-stranded polynucleotides. Finally, interacting energies of a regular duplex and the alternative structures were compared. It was found that the type II model is energetically more favourable than the rest.

Base Sequence↗
PubMed1979

Increased binding of ethidium bromide to polynucleotide duplexes containing mismatched bases.

The binding of the intercalating dye ethidium bromide to a series of synthetic polynucleotide duplexes containing varying concentrations of mismatched bases has been measured by fluorescence titration. The dye binds more strongly to duplexes with mismatches, the estimated increase in affinity being twenty-fold for the series of molecules poly (I).poly (C,Ax) with x denoting the mole fraction of mismatched A residues in the C strand. The results are consistent with one requirement of the Streisinger model for frameshift mutagenesis, namely that frameshifting agents can function by stablizing mismatched transient intermediates in DNA.

Base Composition↗
PubMed1980

Interaction of polynucleotides with natural and model membranes.

Polynucleotides adsorb on natural and model phospholipid membranes in the presence of Mg2+-cations. Adsorption of nucleic acids on membranes results in a considerable change of their secondary structure. The presence of model phosphatidylcholine membranes greatly stimulates the rate of the synthesis of RNA by E. coli RNA-polymerase on DNA template.

Animals↗
PubMed2004

Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase.

T4 phage polynucleotide kinase (PNK) displays 5'-hydroxyl kinase, 3'-phosphatase and 2',3'-cyclic phosphodiesterase activities. The enzyme phosphorylates the 5' hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5' ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5'-GT DNA end than for a 5'-TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.

Bacteriophage T4↗
PubMed2006

The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks.

The dual function mammalian DNA repair enzyme, polynucleotide kinase (PNK), facilitates strand break repair through catalysis of 5'-hydroxyl phosphorylation and 3'-phosphate dephosphorylation. We have examined the relative activities of the kinase and phosphatase functions of PNK using a novel assay, which allows the simultaneous characterization of both activities in processing nicks and gaps containing both 3'-phosphate and 5'-hydroxyl. Under multiple turnover conditions the phosphatase activity of the purified enzyme is significantly more active than its kinase activity. Consistent with this result, phosphorylation of the 5'-hydroxyl is rate limiting in cell extract mediated-repair of a nicked substrate. On characterizing the effects of individually mutating the two active sites of PNK we find that while site-directed mutagenesis of the kinase domain of PNK does not affect its phosphatase activity, disruption of the phosphatase domain also abrogates kinase function. This loss of kinase function requires the presence of a 3'-phosphate, but it need not be present in the same strand break as the 5'-hydroxyl. PNK preferentially binds 3'-phosphorylated substrates and DNA binding to the phosphatase domain blocks further DNA binding by the kinase domain.

Binding Sites↗
PubMed1992

Structural analysis of nucleic acids by precise denaturing gradient gel electrophoresis: II. Applications to the analysis of subtle and drastic mobility changes of oligo- and polynucleotides.

Precise denaturing gradient gel electrophoresis was effectively applied to various kinds of oligo- and polynucleotides. The analyses on oligonucleotides revealed that every oligonucleotide has its own characteristic normalized mobility profile (NMP), which can be used to identify, characterize and classify the molecules. The precise system also enabled us to obtain unequivocally the mobility transitions corresponding to the melting of hairpin structures of oligonucleotides, single-stranded (ss) DNAs, and RNAs. Another application to co-migration and separate migration experiments demonstrated that there were significant binding interactions between two species of ss molecules of similar mobility, even when they have little complementarity with each other. When the precise temperature gradient gel electrophoresis was applied to double-stranded DNAs, it could be confirmed with high reliability that the mobility transitions observed correspond to cooperative meltings and strand dissociations. Through these experiments, mu m, a parameter defined as a mobility transition point, was shown to be effective to deal with those phenomena quantitatively.

Base Sequence↗