The Kinetics of the Interaction of a Helix-Destabilizing Protein from Roe-Deer Liver with DNA and the Influence of Phosphorylation (original) (raw)
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Nucleic Acids Research, 2009
Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly(dA) and oligo(dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo(dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase. The chromatin 3'phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo(dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which ...
Impact of Pyrophosphate and O -Ethyl-Substituted Pyrophosphate Groups on DNA Structure
The Journal of Physical Chemistry B, 2007
Design of the novel DNA probes to inhibit specific repair pathways is important for basic science applications and for use as therapeutic agents. As shown previously, single pyrophosphate (PP) and O-ethyl-substituted pyrophosphate (SPP) modifications can inhibit the DNA glycosylase activities on damaged DNA. To understand structural basis of this inhibition, the influence of the PP and SPP internucleotide groups on the helical parameters and geometry of a double-stranded DNA was studied by using molecular modeling tools including molecular dynamics and quantum mechanical-molecular mechanical (QM/MM) approaches. Native and locally modified PP-and SPP-containing DNA duplexes of dodecanucleotide d(C1G2C3G4A5A6T7T8C9G10C11G12) were simulated in aqueous solution. The energies and forces were computed by using the PBE0/6-31+G** approach in the QM part and the AMBER force field parameters in the MM part. Analysis of the local base-pair helical parameters, internucleotide distances and overall global structure at the located stationary points revealed a close similarity of the initial and modified duplexes, with only torsion angles of the main chain being altered in the vicinity of introduced chemical modification. Results show that the PP and SPP groups are built into a helix structure without elongation of the internucleotide distance due to flipping-out of phosphate group from the sugar-phosphate backbone. The mechanism of such embedding has only a minor impact on the base pairs stacking and Watson-Crick interactions. Biochemical studies revealed that the PP and SPP groups immediately 5', but not 3', to the 8-oxoguanosine (8oxodG) inhibit translesion synthesis by a DNA polymerase in vitro. These results suggest that subtle perturbations of the DNA backbone conformation influence processing of base lesions.
Biochemical and Biophysical Research Communications, 1992
Purified type II (0) and type III (CL) protein kinase C phosphorylates highly purified polyADPribose polymerase irk vitro whereby 2 mols of phosphate are transferred from ATP to serine and threonine residues present in the 36 and 56 kDa polypeptide domains of the polymerase protein, Calf thymus DNA was a non-competitive inhibitor of the protein kinase C catalyzed phosphorylation of polyADP-ribose polymerase. Coincidental with the phosphorylation of the protein the polymerase activity and DNA binding capacity of polyADP-ribose polymerase were inhibited. These in vitro findings may have possible cell biological significance in cellular signal transduction. 0 1992 Academic Press. Inc. Poly ADP-ribose polymerase (PARP) is a highly abundant nonhistone nuclear protein of higher eukaryotes (1,2) which exhibits a cellular differentiation-dependent variation in catalytic activities, consisting of NAD-glycohydrolase and ADP-ribose polymerase (3). Besides enzymatic activities, PARP possesses significant colligative properties towards DNA termini and certain internal DNA structures (4,5,6,7) as well as towards a number of DNA binding nuclear proteins which can serve as polyADP-ribose acceptors (S-13). The catalytic activity of PARP is enhanced more than lOO-fold by DNA possessing free termini and this activation is relatively nonspecific, i.e. various fragmented DNAs can serve as "coenzymes". However even with synthetic octadeoxyribonucleotides there is a secondary influence of base sequences on catalytic activation (14). In contrast to DNA termini, circular DNAs also bind PARP, an association that results in
Involvement of basic amino acids in the activity of a nucleic acid helix-destabilizing protein
Biochimica et biophysica acta, 1981
Under conditions of low ionic strength, ribonuclease A, which binds more tightly to single- than to double-stranded DNA, lowers the melting temperature of DNA helices (Jensen and von Hippel (1976) J. Biol. Chem. 251, 7198-7214). The effects of chemical modification of lysine and arginine residues on the helix-destabilizing properties of this protein have been examined. Removal of the positive charge on the lysine epsilon-amino group, either by maleylation or acetylation, destroys the ability of RNAase A to lower the Tm of poly[d(A-T)]. However, reductive alkylation of these residues, which has not effect on charge, yields derivatives which lower the Tm by only about one-half that seen with unmodified controls. Phenylglyoxalation of arginines can largely remove the Tm-depressing activity of RNAase A. RNAase S, which is produced by cleavage of RNAase A between amino acids 20 and 21, possesses DNA helix-destabilizing activity comparable to that of the parent protein, whereas S-protein ...
Proceedings of the National Academy of Sciences, 1978
The reaction of chloroacetaldehyde with adenine bases in DNA to give a fluorescent product was used to study the availability to intermolecular reaction of positions 1 and 6 of adenine in DNA complexed with calf thymus DNA helix-destabilizing protein. No inhibition of this reaction was observed when heat-denatured DNA was complexed with the protein at a protein/DNA weight ratio of 10:1, compared to free DNA. On the contrary, the same reaction was inhibited markedly for denatured DNA in the presence of calf thymus histone Hi at protein/DNA weight ratio of 2:1. Furthermore, the exchange rate for hydrogens of amino and imide groups of DNA bases in DNA strands with deuterium in the solvent was totally unaffected upon complexing of DNA with the DNA helix-destabilizing protein as examined by stoppd-flow ultraviolet spectroscopy. These results indicate that the DNA helix-destabilizing protein forms a complex with single-stranded DNA, leaving DNA bases uncovered by the protein. The fluorescence intensity of DNA pretreated with chloroacetaldehyde was amplified by nearly 3fold upon addition of the DNA helixdestabilizing protein. The possibility of "unstacking" of DNA bases induced by the protein is discussed.
Journal of Biomolecular Structure and Dynamics, 2016
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3′-end-blocking modifications, possesses DNA and RNA 3′-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3′-end of DNA have been used to prevent 3′-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3′-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single-and double-stranded DNA substrates shows that the rates of the 3′-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3′-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.
European Journal of Biochemistry, 1999
The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA±DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA±DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5 H -O-3thiotriphosphate or guanosine 5 H -O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA±DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex. q FEBS 1999 Effect of nucleotide cofactors on RecA (Eur. J. Biochem. 262) 89
Modulation of DNA polymerases α, δ and ε by lactate dehydrogenase and 3-phosphoglycerate kinase
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1998
. Literature documents that glycolytic enzymes among them lactate dehydrogenase and 3-phosphoglycerate kinase can reside in nuclei of mammalian cells and exert functions in DNA replication, transcription and DNA repair, in addition to their role as catalysts in the cytoplasm. Transfer of glycolytic enzymes to cell nuclei requires modification, for example Ž . phosphorylation. We studied the effects of phosphorylated lactate dehydrogenase and 3-phosphoglycerate kinase on i Ž . UV-induced DNA repair, using permeabilized human fibroblasts, and ii in vitro DNA synthesis catalyzed by purified Ž . DNA polymerases a, d , and´from proliferating rat liver. i Phosphorylated lactate dehydrogenase stimulated UV-induced DNA repair synthesis in normal fibroblasts in a dose-dependent manner; the unphosphorylated enzyme slightly inhibited. In repair-deficient xeroderma pigmentosum fibroblasts reparative synthesis was not enhanced whether lactate dehydrogenase was phosphorylated or not, indicating that reparative DNA synthesis must be possible in order to be Ž . stimulated. ii Activity of purified DNA polymerases a , d , and´was differentially stimulated or inhibited, according to the phosphorylation status of lactate dehydrogenase. DNA polymerases were also modulated by 3-phosphoglycerate kinase, Ž . depending on the primer-templates used which were gapped DNA mimicking a repair mode of DNA synthesis or Ž . single-stranded M13 DNA representing the replicative mode of DNA synthesis . Since glycolytic enzymes in cell nuclei retain binding ability for their cofactors, cytoplasmic substrates and inhibitors, a regulatory linkage might exist between the energy state of a cell and its replicative and reparative functions. q 1998 Elsevier Science B.V.
Chemico-Biological Interactions, 1986
Various trialkyl phosphates were investigated as model compounds for DNA-phosphotriesters for their stability in neutral or alkaline conditions. The results show that phosphotriesters were highly stable even at strongly alkaline pH, with the exception of diethyl 2-hydroxyethyl phosphate (DHP). The extreme instability of the latter was found to be due to the 2-hydroxy function. In accordance with earlier interpretations the 2-hydroxyethyl group is proposed to participate in the formation of a highly reactive dioxaphospholane ring intermediate which decays rapidly by hydrolysis. Alkylation of 3'-and 5'-deoxythymidine monophosphates with methyl-or hydroxyethylnitrosourea (MNU, HENU) results in practically exclusive phosphate alkylation. In analogy with the model phosphotriesters, di (2'-deoxythymidine) phosphotriesters generated after reaction with MNU or HENU showed extreme dependence of their stabilities on the nature of the alkyl group transferred to phosphate. Whereas the methyl phosphotriester was highly stable, the corresponding hydroxyethyl analogue showed half lives of decay of <1 min (pH 12.5), 27 rain (pH 9.1) and 60 min (pH 7). Thus the introduction of a 2-hydroxyethyl function into phosphate strongly decreases the stability of the phosphate link of DNA, resulting in DNA single strand breaks, in analogy to RNA phosphotriesters which have been found earlier to be highly unstable because of the presence of the ribose 2'-OHgroup.