Investigation of thermal stability characteristic in family A DNA polymerase - A theoretical study.

DNA polymerases create complementary DNA strands in living cells and are crucial to genome transmission and maintenance. These enzymes possess similar human right-handed folds which contain thumb, fingers, and palm subdomains and contribute to polymerization activities. These enzymes are classified into seven evolutionary families, A, B, C, D, X, Y, and RT, based on amino acid sequence analysis and biochemical characteristics. Family A DNA polymerases exist in an extended range of organisms including mesophilic, thermophilic, and hyper-thermophilic bacteria, participate in DNA replication and repair, and have a broad application in molecular biology and biotechnology. In this study, we attempted to detect factors that play a role in the thermostability properties of this family member despite their remarkable similarities in structure and function. For this purpose, similarities and differences in amino acid sequences, structure, and dynamics of these enzymes have been inspected. Our results demonstrated that thermophilic and hyper-thermophilic enzymes have more charged, aromatic, and polar residues than mesophilic ones and consequently show further electrostatic and cation-pi interactions. In addition, in thermophilic enzymes, aliphatic residues tend to position in buried states more than mesophilic enzymes. These residues within their aliphatic parts increase hydrophobic core packing and therefore enhance the thermostability of these enzymes. Furthermore, a decrease in thermophilic cavities volumes assists in the protein compactness enhancement. Moreover, molecular dynamic simulation results revealed that increasing temperature impacts mesophilic enzymes further than thermophilic ones that reflect on polar and aliphatic residues surface area and hydrogen bonds changes.

Direct fluorescence detection of microRNA based on enzymatically engineered primer extension poly-thymine (EPEPT) reaction using copper nanoparticles as nano-dye.

A new strategy based on enzymatically engineered primer extension poly-thymine (EPEPT) and nanomaterials in situ generation technology is reported for direct detection of microRNA (miRNA) in a fluorescence turn-on format using the sequential and complementary reactions catalyzed by Klenow Fragment exo- (KFexo-) and terminal deoxynucleotidyl transferase (TdTase). The short miRNA can be efficiently converted into long poly-thymine (polyT) sequences, which function as template for in situ formation of fluorescence copper nanoparticles (CuNPs) as nano-dye for detecting miRNA. The polyT-CuNPs can effectively form and emit intense red fluorescence under the 340nm excitation. For the proof of concept, microRNA-21 (miR-21) was selected as the model target to testify this strategy as a versatile assay platform. By directly using miR-21 as the primer, the simple, rapid and sensitive miRNA detection was successfully achieved with a good linearity between 1pM and 1nM and a detection limit of 100fM. Thus, the EPEPT strategy holds great potential in biochemical sensing research as an efficient and universal platform.

Polyprenylated Benzoylphloroglucinols with DNA Polymerase Inhibitory Activity from the Fruits of Garcinia schomburgkiana.

Chemical investigation of the fruits of Garcinia schomburgkiana collected in Vietnam led to the isolation of eight new schomburgkianones, A-H (1-8), four known (9-12) polyprenylated benzoylphloroglucinols, and four known biflavonoids. The structures of these compounds were elucidated by spectroscopic and chemical means. The absolute configuration at C-40 of 1 and 2 was determined by (1)H NMR analyses of their MPA esters. The configuration of the bicyclo[3.3.1]nonane core of the polyprenylated benzoylphloroglucinols was assigned by comparison of their experimental ECD spectra with those of related compounds. The polyprenylated benzoylphloroglucinols exhibited inhibitory activities against mammalian DNA polymerases α and λ, with IC50 values ranging from 5.0 to 8.8 µM. Compounds 1, 2, 4, 5, and 9-11 showed cytotoxic effects against HeLa human cervical cancer cells with median lethal dose values lower than 10 µM.

Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction.

DNA polymerases (pols) are sophisticated protein machines operating in the replication, repair and recombination of genetic material in the complex environment of the cell. DNA pol reactions require at least two divalent metal ions for the phosphodiester bond formation. We explore two understudied roles of metals in pol transactions with emphasis on polα, a crucial enzyme in the initiation of DNA synthesis. We present evidence that the combination of many factors, including the structure of the template/primer, the identity of the metal, the metal turnover in the pol active site, and the influence of the concentration of nucleoside triphosphates, affect DNA pol synthesis. On the poly-dT70 template, the increase of Mg(2+) concentration within the range typically used for pol reactions led to the severe loss of the ability of pol to extend DNA primers and led to a decline in DNA product sizes when extending RNA primers, simulating the effect of "counting" of the number of nucleotides in nascent primers by polα. We suggest that a high Mg(2+) concentration promotes the dynamic formation of unconventional DNA structure(s), thus limiting the apparent processivity of the enzyme. Next, we found that Zn(2+) supported robust polα reactions when the concentration of nucleotides was above the concentration of ions; however, there was only one nucleotide incorporation by the Klenow fragment of DNA pol I. Zn(2+) drastically inhibited polα, but had no effect on Klenow, when Mg(2+) was also present. It is possible that Zn(2+) perturbs metal-mediated transactions in pol active site, for example affecting the step of pyrophosphate removal at the end of each pol cycle necessary for continuation of polymerization.

Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766.

The accuracy of high-fidelity DNA polymerases such as DNA polymerase I (Klenow fragment) is governed by conformational changes early in the reaction pathway that serve as fidelity checkpoints, identifying inappropriate template-nucleotide pairings. The fingers-closing transition (detected by a fluorescence resonance energy transfer-based assay) is the unique outcome of binding a correct incoming nucleotide, both complementary to the templating base and with a deoxyribose (rather than ribose) sugar structure. Complexes with mispaired dNTPs or complementary rNTPs are arrested at an earlier stage, corresponding to a partially closed fingers conformation, in which weak binding of DNA and nucleotide promote dissociation and resampling of the substrate pool. A 2-aminopurine fluorescence probe on the DNA template provides further information about the steps preceding fingers closing. A characteristic 2-aminopurine signal is observed on binding a complementary nucleotide, regardless of whether the sugar is deoxyribose or ribose. However, mispaired dNTPs show entirely different behavior. Thus, a fidelity checkpoint ahead of fingers closing is responsible for distinguishing complementary from noncomplementary nucleotides and routing them toward different outcomes. The E710A mutator polymerase has a defect in the early fidelity checkpoint such that some complementary dNTPs are treated as if they were mispaired. In the Y766A mutant, the early checkpoint functions normally, but some correctly paired dNTPs do not efficiently undergo fingers closing. Thus, both mutator alleles cause a blurring of the distinction between correct and incorrect base pairs and result in a larger fraction of errors passing through the prechemistry fidelity checkpoints.

A New Method of the Visualization of the Double-Stranded Mitochondrial and Nuclear DNA.

The study describes the method of a sensitive detection of double-stranded DNA molecules in situ. It is based on the oxidative attack on the deoxyribose moiety by copper(I) in the presence of oxygen. We have shown previously that the oxidative attack leads to the formation of frequent gaps in DNA. Here we have demonstrated that the gaps can be utilized as the origins for an efficient synthesis of complementary labeled strands by DNA polymerase I and that such enzymatic detection of the double-stranded DNA is a sensitive approach enabling in-situ detection of both the nuclear and mitochondrial genomes in formaldehyde-fixed human cells.

Inhibitory effects of low molecular weight polyphenolics from Inonotus obliquus on human DNA topoisomerase activity and cancer cell proliferation.

Low molecular weight (LMW) polyphenolics containing a polyhydroxylated benzyl moiety are abundant in medicinal plants. In the present study, we report on the activities of seven LMW polyphenolics isolated from Inonotus obliquus, a medicinal mushroom. The isolated compounds included caffeic acid (CA), 3,4-dihydroxybenzalacetone (DBL), gallic acid, syringic acid, protocatechuic acid, 3,4-dihydroxybenzaldehyde and 2,5-dihydroxyterephthalic acid. We analyzed their inhibitory effects on DNA polymerase (pol) and DNA topoisomerase (topo), and their effects on human cancer cell growth. All isolated compounds inhibited human topo II activity; the most potent were DBL and CA, which contain a catechol propanoid moiety. CA and DBL inhibited the activity of human topo I, whereas other compounds had no effect. No compound modulated the activities of 11 mammalian pol species or other DNA metabolic enzymes, including T7 RNA polymerase, mouse IMP dehydrogenase (type II), T4 polynucleotide kinase and bovine deoxyribonuclease I. CA and DBL markedly suppressed the proliferation of human colon HCT116 carcinoma cells with an LD50 of 70.0 and 49.4 µM, respectively, and halted the cell cycle in the G2/M phase. The suppressive effect of these compounds on cancer cell growth correlated with their ability to inhibit topo II. These results suggest that CA- and DBL-dependent decreases in cell proliferation are due to the inhibition of cellular topo II. The mechanism of action of these catechol propanoid compounds and the implication for their use as anticancer agents are discussed.

Biochemical behavior of N-oxidized cytosine and adenine bases in DNA polymerase-mediated primer extension reactions.

To clarify the biochemical behavior of 2'-deoxyribonucleoside 5'-triphosphates and oligodeoxyribonucleotides (ODNs) containing cytosine N-oxide (C(o)) and adenine N-oxide (A(o)), we examined their base recognition ability in DNA duplex formation using melting temperature (T(m)) experiments and their substrate specificity in DNA polymerase-mediated replication. As the result, it was found that the T(m) values of modified DNA-DNA duplexes incorporating 2'-deoxyribonucleoside N-oxide derivatives significantly decreased compared with those of the unmodified duplexes. However, single insertion reactions by DNA polymerases of Klenow fragment (KF) (exo(-)) and Vent (exo(-)) suggested that C(o) and A(o) selectively recognized G and T, respectively. Meanwhile, the kinetic study showed that the incorporation efficiencies of the modified bases were lower than those of natural bases. Ab initio calculations suggest that these modified bases can form the stable base pairs with the original complementary bases. These results indicate that the modified bases usually recognize the original bases as partners for base pairing, except for misrecognition of dATP by the action of KF (exo(-)) toward A(o) on the template, and the primers could be extended on the template DNA. When they misrecognized wrong bases, the chain could not be elongated so that the modified base served as the chain terminator.

Distinct roles of the active-site Mg2+ ligands, Asp882 and Asp705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions.

DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg(2+) ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp(705) and Asp(882), of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp(882); this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp(882) position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp(705) carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg(2+) into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg(2+) concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing.

Host-specific replication of BK virus DNA in mouse cell extracts is independently controlled by DNA polymerase alpha-primase and inhibitory activities.

The activation of the human polyomavirus BK causes polyomavirus-associated nephropathy in immunocompromised humans. Studies of the virus have been restricted since the virus DNA replication is species specific. Cell-based and cell-free DNA replication systems, including the BK virus (BKV) monopolymerase DNA replication system using purified proteins, reproduce the species specificity (28). Therefore, the major host proteins comprising this assay, DNA polymerase alpha-primase (Pol-prim) and replication protein A (RPA), were intensively studied here. We demonstrate that Pol-prim plays a major role in the species specificity of BKV DNA replication. Both large subunits p180 and p68 of the enzyme complex have central functions in modulating the host specificity. Recently, an inhibitory activity of BKV DNA replication was described (C. Mahon, B. Liang, I. Tikhanovich, J. R. Abend, M. J. Imperiale, H. P. Nasheuer, and W. R. Folk, J. Virol. 83:5708-5717, 2009), but neither mouse Pol-prim nor mouse RPA diminishes cell-free BKV DNA replication. However, the inhibition of BKV DNA replication in mouse extracts depends on sequences flanking the core origin. In the presence of human Pol-prim, the inhibitory effect of mouse cell factors is abolished with plasmid DNAs containing the murine polyomavirus early promoter region, whereas the late enhancer region and the core origin are supplied from BKV. Thus, BKV replication is regulated by both Pol-prim, as a core origin species-specific factor, and inhibitory activities, as origin-flanking sequence-dependent factor(s).

DNA helicase activity in purified human RECQL4 protein.

Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3'-5' direction. These results suggest that the hRECQL4 protein exhibits DNA helicase activity.

Inhibitory effect of sulfoquinovosyl diacylglycerol on prokaryotic DNA polymerase I activity and cell growth of Escherichia coli.

We isolated the glycolipids fraction from spinach (Spinacia oleracea L.) and found that the fraction inhibited the activities of prokaryotic DNA polymerase I from Escherichia coli (E. coli) and cell growth of E. coli. The fraction contained mainly three glycolipids, monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG), and purified SQDG inhibited these activities, however, purified MGDG and DGDG had no influence. In the tested strains of E. coli, SQDG inhibited the cell proliferation of the JM109 strain. It could be considered that a SQDG-containing thylakoid membrane in plant chloroplasts might have anti-bacterial activity.

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays.

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiments showed that the elongation temperature of 50 degrees C and the Mg(2+) concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.

Advanced preclinical and clinical trials of natural products and related compounds from marine sources.

The marine environment has proven to be a very rich source of extremely potent compounds that have demonstrated significant activities in anti-tumor, anti-inflammatory, analgesia, immuno-modulation, allergy and anti-viral assays. Although the case can and has been made that the nucleosides such as Ara-A and Ara-C are derived from knowledge gained from investigations of bioactive marine nucleosides, no drug directly from marine sources (whether isolated or by total synthesis) has yet made it to the commercial sector in any human disease. However, as shown in this review, there are now significant numbers of very interesting molecules that have come from marine sources, or have been synthesized as a result of knowledge gained from a prototypical compound, that are either in or approaching Phase III clinical trials in cancer, analgesia and allergy, with a very substantial number of other, quite different potential agents following in their wake, in these and in other diseases.

Inhibition of DNA polymerases and DNA topoisomerase II by triterpenes produced by plant callus.

We found that some triterpene compounds could not only selectively inhibit the activities of mammalian DNA polymerase alpha (pol alpha) and beta (pol beta), but could also potently inhibit DNA topoisomerase II (topo II) [Biochem. J. 350 (2000) 757]. Here, we report that natural triterpenes produced by callus from an ancient Chinese medicinal plant were also inhibitors of the enzymes, and some were more selective than others. The natural triterpenes with a carboxyl group equally inhibited the activities of pol alpha, pol beta, and topo II, while the olide-type triterpenes with a ketone group suppressed the activities of pol beta and topo II, but not pol alpha. The other triterpenes from the callus hardly influenced these enzyme activities. As also described previously [J. Biochem. 130 (2001) 657], pol beta and topo II have a three-dimensionally similar triterpene-binding region, which is a pocket in which specific compounds can insert. The newly found triterpene inhibitors might structure-dependently insert into the pocket, and the pocket structure of each enzyme might, three-dimensionally but slightly, differ among them. The triterpene frames could be used for screening new inhibitors of the enzymes, and computer-simulated drug design using the frame and pocket structure may in theory be a possible approach to develop new inhibitors.

Enzymatic repair of selected cross-linked homoduplex molecules enhances nuclear gene rescue from Pompeii and Herculaneum remains.

Ancient DNA (aDNA) samples extracted from the bone remains of six equids buried by the Vesuvius eruption in 79 AD were investigated to test pre-amplification and enzymatic repair procedures designed to enhance the rescue of nuclear genes. The extracts, which proved all positive for Equidae mtDNA amplification, proved positive only four times out of 18 when tested for single-copy Equidae nuclear genes (epsilon globin, p53 and gamma interferon). Pre-amplification did not change the number of retrieved aDNA sequences but 10 times out of 14 enzymatic repair restored the amplifiability of the genes analysed, proving that repair increases the rate of successful rescue from 22 to alpha(lambda)mu(omicron)sigma(tau) 80%. These findings support the hypothesis that some of these cross-linked aDNA molecules, which are not completely separated when DNA is extracted under denaturing conditions, become homoduplex substrates for Pol I and/or T4 ligase action upon renaturation. aDNA authenticity is proved by the homology of the nucleotide sequences of loci tested to the corresponding modern Equidae sequences. Data also indicate that cross-linked homoduplex molecules selected by denaturation of the extract are repaired without any chimera formation. The general features of aDNA amplification with and without denaturation and enzymatic repair are discussed.

A human DNA helicase homologous to the DNA cross-link sensitivity protein Mus308.

Repair of DNA interstrand cross-links is a challenging problem for cells. Many human gene products influence sensitivity to DNA cross-linking agents, but the mechanisms of cross-link repair are unknown. In Drosophila melanogaster, the mus308 mutation leads to marked sensitivity to DNA cross-linking agents. The C-terminal portion of the Mus308 polypeptide encodes a DNA polymerase, whereas a putative DNA helicase is encoded by the N-terminal portion. As a step toward isolating proteins involved in DNA cross-link repair, we searched for mammalian genes similar to the DNA helicase portion of Mus308. Human and mouse homologs were isolated from cDNA expression libraries and designated HEL308. Human HEL308 is on chromosome 4q21 and encodes a polypeptide of 1101 amino acids. The protein was expressed in insect cells and purified. HEL308 is a single-stranded DNA-dependent ATPase and DNA helicase. Mutation of a highly conserved lysine to methionine in helicase domain I eliminated both activities. The protein readily displaces 20- to 40-mer duplex oligonucleotides. Displacement of longer substrates was less efficient but was stimulated by the single-stranded DNA-binding protein RPA. Activity was supported by ATP or dATP but not other nucleotide triphosphates. The enzyme translocates on DNA with 3' to 5' polarity and behaves as a multimer upon gel filtration.

Direct monitoring kinetic studies of DNA polymerase reactions on a DNA-immobilized quartz-crystal microbalance.

Catalytic reactions of DNA polymerase I from E. coli (Klenow fragment, KF) were monitored directly with a template/primer (40/25- or 75/25-mer)-immobilized 27-MHz quartz-crystal microbalance (QCM). The 27-MHz QCM is a very sensitive mass-measuring device in aqueous solution, as the frequency decreases linearly with increasing mass on the QCM electrode at the nanogram level. Three steps in polymerase reactions which include 1) binding of DNA polymerase to the primer on the QCM (mass increase); 2) elongation of complementary nucleotides along the template (mass increase); and 3) release of the enzyme from the completely polymerized DNA (mass decrease), could be monitored continuously from the time dependencies of QCM frequency changes. The binding constant (Ka) of KF to the template/primer DNA was 10(8)M(-1) (k(on) = 10(5)M(-1)s(-1) and k(off)= 10(-3)s(-1)), and decreased to 10(6)M(-1) (k'on = 10(4)M(-1)s(-1) and k'off = 10(-2)s(-1)) for completely polymerized DNA. This is due to the 10-fold decrease in binding rate constant (k(on)) and 10-fold increase in dissociation rate constant (k(off)) for completed DNA strands. Ka values depended slightly on the template and primer sequences. The kinetic parameters in the elongation process (k(cat) and Km) depended only slightly on the DNA sequences. The repair process during the elongation catalyzed by KF could also be monitored in real time as QCM frequency changes.

Contacts between the 5' nuclease of DNA polymerase I and its DNA substrate.

The 5' nuclease of DNA polymerase I (Pol I) of Escherichia coli is a member of an important class of prokaryotic and eukaryotic nucleases, involved in DNA replication and repair, with specificity for the junction between single-stranded and duplex DNA. We have investigated the interaction of the 5' nuclease domain with DNA substrates from the standpoint of both the protein and the DNA. Phosphate ethylation interference showed that the nuclease binds to the nucleotides immediately surrounding the cleavage site and also contacts the complementary strand one-half turn away, indicating that contacts are made to one face only of the duplex portion of the DNA substrate. Phosphodiester contacts were investigated further using DNA substrates carrying unique methylphosphonate substitutions, together with mutations in the 5' nuclease. These experiments suggested that two highly conserved basic residues, Lys(78) and Arg(81), are close to the phosphodiester immediately 5' to the cleavage site, while a third highly conserved residue, Arg(20), may interact with the phosphodiester 3' to the cleavage site. Our results provide strong support for a DNA binding model proposed for the related exonuclease from bacteriophage T5, in which the conserved basic residues mentioned above define the two ends of a helical arch that forms part of the single-stranded DNA-binding region. The nine highly conserved carboxylates in the active site region appear to play a relatively minor role in substrate binding, although they are crucial for catalysis. In addition to binding the DNA backbone around the cleavage point, the 5' nuclease also has a binding site for one or two frayed bases at the 3' end of an upstream primer strand. In agreement with work in related systems, 5' nuclease cleavage is blocked by duplex DNA in the 5' tail, but the enzyme is quite tolerant of abasic DNA or polarity reversal within the 5' tail.

Incoming nucleotide binds to Klenow ternary complex leading to stable physical sequestration of preceding dNTP on DNA.

Klenow-DNA complex is known to undergo a rate-limiting, protein conformational transition from an 'open' to 'closed' state, upon binding of the 'correct' dNTP at the active site. In the 'closed' state, Mg(2+) mediates a rapid chemical step involving nucleophilic displacement of pyrophosphate by the 3' hydroxyl of the primer terminus. The enzyme returns to the 'open' state upon the release of PPi and translocation permits the next round of reaction. To determine whether Klenow can translocate to the next site on the addition of the next dNTP, without the preceding chemical step, we studied the ternary complex (Klenow-DNA-dNTP) in the absence of Mg(2+). While the ternary complex is proficient in chemical addition of dNTPs in Mg(2+), as revealed by primer extensions, the same in Mg(2+)-deficient conditions lead to non-covalent (physical) sequestration of first two 'correct' dNTPs in the ternary complex. Moreover, the second dNTP traps the first one in the DNA-helix of the ternary complex. Such a dNTP-DNA complex is found to be stable even after the dissociation of KLENOW: This reveals the novel state of the dNTP-DNA complex where the complementary base is stacked in a DNA-helix non-covalently, without the phosphodiester linkage. Further, shuttling of the DNA between the polymerase and the exonuclease site mediates the release of such a DNA complex. Interestingly, Klenow in such a Mg(2+)-deficient ternary complex exhibits a 'closed' conformation.