Presence Of dNTP Research Articles

DNTP-Dependent Conformational Transitions in the Fingers Subdomain of Klentaq1

DNA polymerases are responsible for the accurate replication of DNA. Kinetic studies indicate the requirement for multiple intermediate enzyme conformations in this process. Structural studies show a large conformational change in the fingers subdomain of DNA polymerase on binding of a correct dNTP. Using single molecule FRET we show that the conformational transition affecting the fingers subdomain also takes place in the apo and DNA-bound forms of the enzyme. In addition a third conformation is observed which is occupied in the presence of dNTP alone, and in the presence of a non base pairing dNTPs. The relative proportions of the identified states are altered dramatically depending on substrate. Binding of the correct nucleotide displaces this equilibrium dramatically towards the closed form, while binding of an incorrect nucleotide favors a more open conformation. The results suggest that the closed state is by design less energetically favored, providing a thermodynamic brake on incorrect nucleotide insertion.

A Mg2+‐induced conformational switch rendering a competent DNA polymerase catalytic complex

The structural and dynamical changes occurring before nucleotide addition were studied using molecular dynamics (MD) simulations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexes containing one or two Mg2+ ions in the presence of dNTP. Our models revealed that the formation of a catalytically competent DNA polymerase complex required subtle rearrangements at the catalytic site A, which occurred only when an Mg2+ ion was bound. This model has been validated using pre-steady-state kinetics to show that free Mg2+ is necessary to obtain a catalytically competent polymerase. Kinetic studies carried out with Be2+ as a cofactor permitted the functional discrimination between metal sites A and B. At low concentrations, Be2+ increased the catalytic efficiency of the polymerase, while at higher concentrations, it competed with Mg2+ for binding to site A, and inhibited DNA polymerization. In agreement with experimental data, MD simulations revealed that the catalytic attack distance between the 3-OH of the primer and the phosphorus in complexes containing Be2+ instead of Mg2+ at site A was above 4.5 A. Our findings provide a detailed description of the mechanism of DNA polymerization and should be helpful to understand the molecular basis of DNA replication fidelity.

Single Stranded Polydeoxyribonucleotides Compete with the Incorporation of [3H] Thymidine or [3H]dAMP into DNA.

Defibrotide, the sodium salt of a single-stranded polydeoxyribonucleotide is prepared by a controlled depolymerisation of deoxyribonucleic acid (DNA) which is obtained from mammalian organs. In previous studies it was shown to mediate immunosuppressive effects (Ferraresso et al. 1993)1.In the current survey we investigated whether randomly chemically synthesized single-stranded polydeoxyribonucleotides of different length and composition could provide similar effects. For this purpose, purified T-cells were stimulated in the presence of dNTP's or single-stranded polydeoxyribonucleotides with irradiated, allogeneic PBMC's, PHA or anti-CD3/CD28 Dynabeads. Cellular proliferation was assessed by incorporation of tritium-labelled thymidine ([3H]thymidine), respectively [3H]dAMP or by staining with CFSE (carboxyfluorescein succinimidyl ester). After 72h or 120h of incubation, the incorporation of [3H]thymidine, or [3H]dAMP as well as the CFSE distribution was assessed. Cell viability was measured by trypan blue exclusion. T-cell activation was measured after 72h by quantifying the number of CD3+ T-cells expressing the activation markers CD25 and CD69. Cellular uptake of fluorochrome-labelled oligos was detected by fluorescence microscopy using an AxioCam HR and visualized by AxioVison. Each experiment was performed at least three times. Polydeoxyribonucleotides of different length, composition or concentrations (up to 5mM) did not cause cytotoxic effects to lymphocytes. But the incorporation of [3H]thymidine or [3H]dAMP was competed by polydeoxyribonucleotides. These effects were found to be dependent on length, concentration and base-composition of the nucleic acids. The proliferative capacity of the T-cells, as assessed by CFSE-staining, seemed to be unaffected. In context with data obtained by fluorescence microscopy, we hypothesise that there might be a cellular uptake of the polydeoxyribonucleotides followed by a subsequent cytoplasmatic degradation and a intracellular reutilisation via the salvage pathway for synthesis of nucleotides. The standard approach to detect cellular proliferation by incorporation of tritium-labelled nucleotides or derivatives is not useful to assess changes in cellular metabolism or proliferation in context with polydeoxyribonucleotides. Moreover, and that might be even more important, treatment approaches using nucleoside analogues like fludarabine, cytarabine e.g. in context with polydeoxyribonucleotides might be critical and diminish the efficacy of these drugs. Further experiments are necessary to elucidate the precise mechanism of these effects.

Phototriggered Formation and Repair of DNA Containing a Site-Specific Single Strand Break of the Type Produced by Ionizing Radiation or AP Lyase Activity

DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage. One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates. Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem. We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector. To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification. The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light. In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E. coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E. coli endonuclease IV. Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I. This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps. In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and multiply damaged sites that might otherwise be difficult to prepare by other methods due to their lability.

Incorporation of cytosine arabinoside monophosphate into DNA at internucleotide linkages by human DNA polymerase alpha

The incorporation of cytosine arabinoside monophosphate (araCMP) into DNA at internucleotide linkages by DNA polymerase alpha (DNA pol alpha) has been investigated by using oligonucleotide primed DNA templates. The products of reactions catalyzed by DNA pol alpha in vitro were analyzed on polyacrylamide gels to measure insertion of araCMP, extension from an araCMP 3' terminus, and binding of the enzyme to an araCMP 3' terminus. The results show that insertion of araCMP opposite dGMP in the DNA template is about 3-fold less efficient than insertion of dCMP. Extension from an araCMP 3' terminus by addition of the next complementary nucleotide is approximately 2000-fold less efficient than extension from a correctly base-paired 3' terminus. In the absence of the second substrate, dNTP, DNA pol alpha binds with approximately equal affinities to DNA templates that contain oligonucleotide primers with araCMP or dCMP positioned at the 3' terminus. In the presence of dNTP, the enzyme extends the araCMP 3' terminus or dissociates, but it is not trapped at the araCMP 3' terminus in a nonproductive ternary complex as is observed at the ddCMP 3' terminus. To determine if slow phosphodiester bond formation contributes to the observed extension rate from the araCMP 3' terminus by DNA pol alpha, oligonucleotide primers with araCMP positioned at the 3' terminus were elongated by addition of the alpha-phosphorothioate analogue of the next complementary nucleotide. The rate of extension from araCMP by addition of 2'-deoxyadenosine 5'-O-phosphorothioate (dAMP alpha S) was 6-fold slower than by addition of dAMP, indicating that bond formation is partially rate limiting in the extension reaction. Thus, inefficient extension from the araCMP 3' terminus is the major determinant contributing to the low incorporation frequency of araCMP into DNA by DNA pol alpha, and this inefficiency can be attributed, in part, to slower phosphodiester bond formation at the araCMP 3' terminus.

Active-site modification of mammalian DNA polymerase .beta. with pyridoxal 5'-phosphate: mechanism of inhibition and identification of lysine 71 in the deoxynucleoside triphosphate binding pocket

Pyridoxal 5'-phosphate is a potent inhibitor of the DNA polymerase activity of recombinant rat DNA polymerase beta. Kinetic studies indicate that the mechanism of PLP inhibition is complex. In a lower range of PLP concentration, inhibition is competitive with respect to substrate dNTP, whereas at higher levels of PLP several forms of enzyme combine with PLP and are involved in the overall inhibition, and a possible model for these interactions during the catalytic process is suggested. Reduction of the PLP-treated enzyme with sodium [3H]borohydride results in covalent incorporation of about 4 mol of PLP/mol of enzyme, and the modified enzyme is not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocks incorporation of 1 mol of PLP/mol of enzyme, and the enzyme so modified is almost fully active. This protective effect is not observed in the absence of template-primer. Tryptic peptide mapping of the PLP-modified enzyme reveals four major sites of modification. Of these four sites, only one is protected by dNTP from pyridoxylation. Sequence analysis of the tryptic peptide corresponding to the protected site reveals that it spans residues 68-80 in the amino acid sequence of the enzyme, with Lys 71 as the site of pyridoxylation. These results indicate that Lys 71 is at or near the binding pocket for the dNTP substrate.

Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (PLP) is an inhibitor of DNA polymerase activity of Escherichia coli DNA polymerase I large fragment. Kinetic studies indicated that overall PLP inhibition was noncompetitive with respect to dNTP, and Hill plot analysis revealed that two molecules of PLP were involved in the inhibition. Reduction of the PLP-treated enzyme with sodium [3H]borohydride resulted in covalent incorporation of 3 mol of PLP/mol of enzyme. This incorporation was at lysine residues exclusively, and the PLP-modified enzyme was not capable of DNA polymerase activity. The presence of dNTP during the modification reaction blocked the incorporation of 1 mol of PLP/mol of enzyme. Similar results were obtained in the presence or absence of template-primer. These data indicate that a PLP target lysine is in or around a dNTP binding site that is essential for polymerase activity and that this binding site is functional in the absence of template-primer. The enzyme modified in the presence of dNTP, containing 2 mol of PLP/mol of enzyme, was capable of DNA polymerase activity but was unable to conduct elongation of product molecules beyond a short oligonucleotide length.

The effect of ATP on the incorporation of deoxyribonucleoside triphosphates by Escherichia coli DNA polymerase I

The effect of ATP on Escherichia coli DNA polymerase I has been investigated as a function of the concentration of substrates and divalent metal ions in the presence of activated DNA as template. At saturating Mg 2+ concentration 1.5 mM ATP stimulated 2.5-times the incorporation of [ 3H]dGTP when only one substrate (dGTP) was present, and had no significant effect in the presence of all four dNTPs, whereas under similar conditions, a saturating concentration of dATP increased the reaction rate only 1.5-times. At optimal Mn 2+ concentrations ATP also showed a similarly marked effect only in the case when one substrate (dGTP) was present in the reaction. The optimal concentration of Mn 2+ was shifted by ATP to higher concentrations both in the presence of one and of all four substrates. ATP did not influence the apparent K m for dGTP, while V was increased by a factor of about 2.5. The possible presence of dNTP in ATP, as inpurity, was ruled out by isotope dilution analysis. Thus, ATP stimulated the polymerization reaction only under limited conditions, i.e., when one substrate was present in the reaction.

Mechanism of 3' to 5' exonuclease associated with phage T5-induced DNA polymerase: processiveness and template specificity.

T5-induced DNA polymerase has an associated 3' to 5' exonuclease activity. Both single-stranded and duplex DNA are hydrolyzed by this enzyme in a quasi-processive manner. This is indicated by the results of polymer-challenge experiments utilizing product analysis techniques. Due to the quasi-processive mode of hydrolysis, the kinetics of label release from the 3'-terminally labeled oligonucleotide substrates, annealed to complementary homopolymers, show an initial high rate of hydrolysis. In the case of both single-stranded and duplex DNA substrates, hydrolysis seems to continue, at best, up to the point where the enzyme is five or six nucleotides away from the 5-end. The enzyme carries out mismatch repair, as evidenced by experiments with primer molecules containing improper base residues at the 3'-OH terminus. Control experiments with complementary base residues at the 3'-end indicate that extensive removal of terminal residue takes place in the presence of dNTP's only when such residues are "improper" in the Watson-Crick sense.