Discrimination against the cytosine analog tC by Escherichia coli DNA polymerase IV DinB

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson-Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.     

Significant impact of divalent metal ions on the fidelity,sugar selectivity,and drug incorporation efficiency of human PrimPol

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn²⁺-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn²⁺, rather than Mg²⁺, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979 nM in the presence of Mn²⁺ and Mg²⁺, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn²⁺ than with Mg²⁺. Notably, the substitution fidelity of PrimPol in the presence of Mn²⁺ was determined to be in the range of 3.4 × 10⁻² to 3.8 × 10⁻¹, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57–1800 with Mn²⁺ and 150–4500 with Mg²⁺, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).     

Interactions of DNA with divalent metal ions. II. Proton relaxation enhancement studies

The extent and modes of binding of the divalent metal ions Mn²⁺ and Co²⁺ to DNA and the effects of salt on the binding have been studied by measurements of the effects of these paramagnetic metal ions on the longitudinal and transverse relaxation rates of the protons of the solvent water molecules, a technique that is sensitive to overall binding. The number of water molecules coordinated to the DNA–bound Mn²⁺ and Co²⁺ is found to be between five and six, and the electron spin relaxation times and the electron-nuclear hyperfine constants associated with Mn²⁺ and Co²⁺ are little or not affected by the binding. These observations indicate little disturbance of the hydration sphere of Mn²⁺ and Co²⁺ upon binding to DNA. An average 2–3-fold reduction in the exchange rate of the water of hydration of the bound metal ions and an order-of-magnitude increase in their rotational correlation time are attributed to hydrogen-bond formation with the DNA. The binding constants of Mn²⁺ to DNA, at metal concentrations approaching zero, are found to be inversely proportional to the second power of the salt concentration, in agreement with the predictions of Manning's polyelectrolyte theory. A remarkable quantitative agreement with the polyelectrolyte theory is also obtained for the anticooperativity in the binding of Mn²⁺ to DNA, although the experimental results can be well accounted for by another simple electrostatic model. The various modes of binding of divalent metal ions to DNA are discussed.     

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.     

Ruthenium red as a carrier of electrons between external NADH and cytochrome c in rat liver mitochondria.

We have determined that Co²⁺, Ni²⁺ or Zn²⁺ may substitute for Mg²⁺ during DNA synthesis with $\text{E.}\text{coli}$ DNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerase from avian myeloblastosis virus (AMV). In addition, the frequency of non-complementary nucleotide incorporation using AMV DNA polymerase was increased using Co²⁺ or Mn²⁺ as the metal activator. These results suggest that the fidelity of DNA synthesis may be influenced by the metal activator used during catalysis.     

Interactions of DNA with divalent metal ions. I. 31P-nmr studies

³¹P-nmr has been used to investigate the specific interaction of three divalent metal ions, Mg²⁺, Mn²⁺, and Co⁺², with the phosphate groups of DNA. Mg²⁺ is found to have no significant effect on any of the ³¹P-nmr parameters (chemical shift, line-width, T₁, T₂, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn²⁺ and Co²⁺, on the other hand, significantly change the ³¹P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn²⁺ and Co²⁺, respectively, corresponding to only 15 ± 5% of the total bound Mn²⁺ and Co²⁺ being directly coordinated to the phosphate groups (inner-sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer-sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the ³¹P relaxation rates of DNA in the absence and presence of Mn²⁺ and Co²⁺ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two-step model was used in the analysis of the kinetic data. The lifetimes of the inner-sphere complexes are 3 × 10^?7 and 1.4 × 10^?5 s for Mn²⁺ and Co²⁺, respectively. The rates of formation of the inner-sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate-determining step in the formation of the inner-sphere complexes. Competition experiments demonstrate that the binding of Mg²⁺ ions is 3–4 times weaker than the binding of either Mn²⁺ or Co²⁺. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (～15%), the higher binding strength of Mn²⁺ and Co²⁺ may be attributed either to base binding or to formation of stronger outer-sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner-sphere phosphate binding increases. In the presence of very high levels of Mg²⁺ (e.g., 3.1M), the inner-sphere ? outer-sphere equilibrium is shifted toward ～100% inner-sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.     

Metal cations as synaptosomal calcium blockers in studies with Fura-2

Metal ions are often used to block calcium channels in various tissues, including synaptosomes. In the present study, Fura-2 was used to determine the effectiveness of various metal ions as calcium channel blockers in rat brain synaptosomes in vitro. In buffer solutions, La³⁺ and Cd²⁺ increased the Fura-2 fluorescence in a manner similar to Ca²⁺. Ni²⁺ and Mn²⁺ appeared to be fluorescence quenching cations, and Sr²⁺ and Co²⁺ had little effect on the fluorescence of Fura-2. In suspensions of synaptosomes under resting conditions, Cd²⁺, Ni²⁺ and Mn²⁺ were found to be not suitable for use in synaptosome studies. On the other hand, La³⁺ and Co²⁺ had little effect on the Fura-2 fluorescence of resting synaptosomes, and under depolarizing conditions, La³⁺ and Co²⁺ decreased the Fura-2 fluorescence. These resuls, therefore, suggest that La³⁺ and Co²⁺ may be suitable as calcium channel blockers in synaptosome studies.     

Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment

Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5′-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tC^O), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.     

New morphotypes of condensed DNA microparticles formed in PCR with <Emphasis Type="Italic">KlenTaq</Emphasis> and <Emphasis Type="Italic">Taq</Emphasis> polymerases and plasmid DNA as a template

The specimens of DNA microparticles formed during PCR amplification of IS-elements ISAfe1 and IST2 by KlenTaq or Taq polymerases and plasmid DNA as a template under varying conditions were investigated by electron microscopy. Microparticle yield and morphology were found to depend on the level of synthesis of single-stranded DNA fragments during PCR. The conditions were studied for formation of discs (ellipsoids) several micrometers in diameter and several dozens of nanometers thick, as well as of microparticles of other morphologies, in the course of PCR with Taq polymerase. The structure of the microparticles produced during an asymmetric PCR, i.e., under conditions of low concentration of one of the two primers, was investigated. Morphology of the DNA micro- and nanoparticles was found to depend mainly on the DNA polymerase used in asymmetric PCR. In particular, in the presence of the KlenTaq polymerase, discs or ellipsoids a few dozen nanometers thick were formed, while in the presence of the Taq polymerase, micro- and nanospheres, heterogeneous in size with rugged surfaces, were produced. The effect of Mn²⁺ cations on DNA microparticle morphology was studied. In the presence of Mn²⁺, microparticle morphology changed dramatically; in PCR mixtures containing KlenTaq polymerase supplemented with Mn²⁺, DNA microspheres with fringed surfaces were formed; in the presence of Taq polymerase, microparticles in the form of short, rounded rods were produced. In light of these data, the molecular mechanism of micro- and nanoparticle formation in the course of PCR is discussed.     

Transition metal ions and selenite modulate the methylation of arsenite by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT)

This report demonstrates that transition metal ions and selenite affect the arsenite methylation by the recombinant human arsenic (+3 oxidation state) methyltransferase (hAS3MT) in vitro. Co²⁺, Mn²⁺, and Zn²⁺ inhibited the arsenite methylation by hAS3MT in a concentration-dependent manner and the kinetics indicated Co²⁺ and Mn²⁺ to be mixed (competitive and non-competitive) inhibitors while Zn²⁺ to be a competitive inhibitor. However, only a high concentration of Fe²⁺ could restrain the methylation. UV-visible, CD and fluorescence spectroscopy were used to study the interactions between the metal ions above and hAS3MT. Further studies showed that neither superoxide anion nor hydrogen peroxide was involved in the transition metal ion or selenite inhibition of hAS3MT activity. The inhibition of arsenite methylating activity of hAS3MT by selenite was reversed by 2 mM DTT (dithiothreitol) but neither by cysteine nor by β-mercaptoethanol. Whereas, besides DTT, cysteine can also prevent the inhibition of hAS3MT activity by Co²⁺, Mn²⁺, and Zn²⁺. Free Cys residues were involved in the interactions of transition metal ions or selenite with hAS3MT. It is proposed that the inhibitory effect of the ions (Co²⁺, Mn²⁺, and Zn²⁺) or selenite on hAS3MT activity might be via the interactions of them with free Cys residues in hAS3MT to form inactive protein adducts.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

Conformational dynamics of DNA polymerase probed with a novel fluorescent DNA base analogue

DNA polymerases discriminate between correct and incorrect nucleotide substrates during a "nonchemical" step that precedes phosphodiester bond formation in the enzymatic cycle of nucleotide incorporation. Despite the importance of this process in polymerase fidelity, the precise nature of the molecular events involved remains unknown. Here we report a fluorescence resonance energy transfer (FRET) system that monitors conformational changes of a polymerase-DNA complex during selection and binding of nucleotide substrates. This system utilizes the fluorescent base analogue 1,3-diaza-2-oxophenothiazine (tC) as the FRET donor and Alexa-555 (A555) as the acceptor. The tC donor was incorporated within a model DNA primer/template in place of a normal base, adjacent to the primer 3' terminus, while the A555 acceptor was attached to an engineered cysteine residue (C751) located in the fingers subdomain of the Klenow fragment (KF) polymerase. The FRET efficiency increased significantly following binding of a correct nucleotide substrate to the KF-DNA complex, showing that the fingers had closed over the active site. Fluorescence anisotropy titrations utilizing tC as a reporter indicated that the DNA was more tightly bound by the polymerase under these conditions, consistent with the formation of a closed ternary complex. The rate of the nucleotide-induced conformational transition, measured in stopped-flow FRET experiments, closely matched the rate of correct nucleotide incorporation, measured in rapid quench-flow experiments, indicating that the conformational change was the rate-limiting step in the overall cycle of nucleotide incorporation for the labeled KF-DNA system. Taken together, these results indicate that the FRET system can be used to probe enzyme conformational changes that are linked to the biochemical function of DNA polymerase.     

A new principle for activity measurement of ADP or ATP dependent enzymes: fluorescence quenching of epsilon-ADP and epsilon-ATP by divalent metal ions.

The divalent metal ions Cu²⁺, Co²⁺, Mn²⁺, and Zn²⁺ form complexes with the fluorescent etheno analogs of the adenine nucleotides. The fluorescence intensity is thereby diminished. The binding strength of the metals to etheno-adenosine triphosphate is higher than to etheno-adenosine di- and monophosphate. The quenching effect of the divalent metal ions can be exploited as a simple routine activity measurement for various kinases and phosphohydrolases.     

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His²⁴⁴ or His²⁶⁶ residue. The binding of Zn²⁺ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co²⁺, Ca²⁺, Ni²⁺, Mg²⁺, and Mn²⁺. The strength of Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺ and Ni²⁺ binding to the site close to His²⁴⁴ is stronger than that of these divalent metal ions binding to the site close to His²⁶⁶.     

怀地黄SRAP扩增体系的建立与引物的筛选

为建立适合怀地黄SRAP-PCR分子标记技术体系,通过单因子实验分别研究了DNA模板浓度、TaqDNA聚合酶浓度、Mg2+浓度、引物浓度以及dNTP浓度对怀地黄SRAP扩增反应的影响,确立了适合怀地黄SRAP最佳反应体系为:在25μL的反应体系中,模板DNA量20ng/25μL、2.5mmol/LMg2+、0.32μmol/L的上下游引物、0.30μmol/L的dNTP以及2.5UTaq酶,并利用确定的体系从88个引物组合中筛选出12对适合怀地黄SRAP-PCR反应的引物。     

早熟砂梨ISSR-PCR反应体系的建立与优化

以我国南方主栽的早熟砂梨品种‘翠冠’Pyrus pyrifolia ‘Cuiguan’为材料,对ISSR技术体系中的模板DNA浓度、Taq DNA聚合酶用量、引物浓度、dNTP浓度、Mg2+浓度、退火温度、PCR循环数等7个主要因素进行优化和筛选,建立了适合早熟砂梨的ISSR-PCR反应体系。最终反应体系为20 μL体系中10×PCR buffer（不含Mg2+）2 μL,模板DNA浓度60 ng,TaqDNA聚合酶0.75 U,引物浓度1 μmol/L,dNTP浓度90 μmol/L,Mg2+浓度2.25 mmol/L。扩增程序为：预变性94 ℃ 5 min,变性94 ℃ 45 s,退火45 s,72 ℃延伸1 min,共42个循环,然后72 ℃再延伸10 min,4 ℃保存,用1.5%琼脂糖凝胶电泳检测多态性。     

Using a Fluorescent Cytosine Analogue tC(o) To Probe the Effect of the Y567 to Ala Substitution on the Preinsertion Steps of dNMP Incorporation by RB69 DNA Polymerase

Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC(o)), to identify preinsertion step(s) altered by NBP flexibility. When tC(o) is the templating base in a wild-type (wt) RB69pol ternary complex, its fluorescence is quenched only in the presence of dGTP. However, with the RB69pol Y567A mutant, the fluorescence of tC(o) is also quenched in the presence of dATP. We determined the crystal structure of the dATP/tC(o)-containing ternary complex of the RB69pol Y567A mutant at 1.9 ? resolution and found that the incoming dATP formed two hydrogen bonds with an imino-tautomerized form of tC(o). Stabilization of the dATP/tC(o) base pair involved movement of the tC(o) backbone sugar into the DNA minor groove and required tilting of the tC(o) tricyclic ring to prevent a steric clash with L561. This structure, together with the pre-steady-state kinetic parameters and dNTP binding affinity, estimated from equilibrium fluorescence titrations, suggested that the flexibility of the NBP, provided by the Y567 to Ala substitution, led to a more favorable forward isomerization step resulting in an increase in dNTP binding affinity.     

Interaction of human DNA polymerase beta with ions of copper, lead, and cadmium

Under appropriate conditions, divalent copper, lead, and cadmium ions significantly inhibit human DNA polymerase β (following accepted convention, the term DNA polymerase β refers to the low-molecular-weight, 3–4 S DNA polymerase of eukaryotic cells) at concentrations below 10^?5m. Each metal showed apparent linear noncompetitive inhibition kinetics with respect to the template primer and the deoxynucleoside triphosphate substrate, indicating that complex formation with these components does not account for the inhibition. Apparently, neither lead nor cadmium inhibit by displacing required zinc atoms from the polymerase. The interaction of the metals with the enzyme can be reversed or prevented by EDTA or by thiol compounds, except that inhibition by cadmium ions can be reversed by monothiols but not by dithiols. The metals probably do not inhibit through reaction with thiol groups since the inhibition is not decreased by pretreating the enzyme with N-ethylmaleimide. Although divalent zinc is moderately inhibitory in manganese activated poly(dT) synthesis on a poly(dA) template, it can fill the requirement for a divalent metal ion and, under the conditions tested, is about 60% as effective as Mn²⁺.     

A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase α from both calf thymus and human lymphoma cells and DNA polymerase β from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG⁴ lesions when Mg²⁺ is the divalent cation. Substitution of Mn²⁺ for Mg²⁺ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase α inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase β is specific, inserting mainly C even in the presence of Mn²⁺. The K_m for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn²⁺ is about 0.5 mm. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg²⁺ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn²⁺. dNTPαS and recA protein increase only the insertion of C.We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.