Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase.

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2'-deoxy-2'-fluoro-ß-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

Partial Purification of a Megadalton DNA Replication Complex by Free Flow Electrophoresis.

We describe a gentle and rapid method to purify the intact multiprotein DNA replication complex using free flow electrophoresis (FFE). In particular, we applied FFE to purify the human cell DNA synthesome, which is a multiprotein complex that is fully competent to carry-out all phases of the DNA replication process in vitro using a plasmid containing the simian virus 40 (SV40) origin of DNA replication and the viral large tumor antigen (T-antigen) protein. The isolated native DNA synthesome can be of use in studying the mechanism by which mammalian DNA replication is carried-out and how anti-cancer drugs disrupt the DNA replication or repair process. Partially purified extracts from HeLa cells were fractionated in a native, liquid based separation by FFE. Dot blot analysis showed co-elution of many proteins identified as part of the DNA synthesome, including proliferating cell nuclear antigen (PCNA), DNA topoisomerase I (topo I), DNA polymerase δ (Pol δ), DNA polymerase ɛ (Pol ɛ), replication protein A (RPA) and replication factor C (RFC). Previously identified DNA synthesome proteins co-eluted with T-antigen dependent and SV40 origin-specific DNA polymerase activity at the same FFE fractions. Native gels show a multiprotein PCNA containing complex migrating with an apparent relative mobility in the megadalton range. When PCNA containing bands were excised from the native gel, mass spectrometric sequencing analysis identified 23 known DNA synthesome associated proteins or protein subunits.

Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site.

The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5'-3' DNA polymerase activity while in absence of 5'-3' exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas.

Realizing directional cloning using sticky ends produced by 3'-5' exonuclease of Klenow fragment.

The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5'-3' polymerase activity can extend the 5' overhanging sticky end to the blunt end, and 3'-5' exonuclease activity can cleave the 3' overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3'-5' exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5' overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost.

Temperature dependence of accuracy of DNA polymerase I from Geobacillus anatolicus.

Klenow-like DNA polymerase I fragment from Geobacillus anatolicus (GF) was cloned and purified. The accuracy of GF was measured in vitro at three different temperatures under single turnover conditions as well as using a forward mutation assay. In pre-steady-state kinetic measurements, when temperature was raised from 22 °C to 50 °C, the rate (k(pol)) for cognate dTTP and non-cognate dATP nucleotide incorporations increased six- and four-fold, respectively, whereas the K(d) for both nucleotide incorporations changed only slightly. As a result, the error frequency was remained constant (∼4 × 10(-4)) over this temperature range. The accuracy of GF was also measured using a forward mutation assay during a single cycle of DNA synthesis of the lacZα complementation gene in M13mp2 DNA. In this assay, which scores various types of replication errors, mutant frequency of GF was 5 × 10(-3) at 72 °C which is four-fold higher than that of 37 °C.

Yeast mitochondrial DNA polymerase is a highly processive single-subunit enzyme.

Polymerase γ is solely responsible for fast and faithful replication of the mitochondrial genome. High processivity of the polymerase γ is often achieved by association of the catalytic subunit with accessory factors that enhance its catalytic activity and/or DNA binding. Here we characterize the intrinsic catalytic activity and processivity of the recombinant catalytic subunit of yeast polymerase γ, the Mip1 protein. We demonstrate that Mip1 can efficiently synthesize DNA stretches of up to several thousand nucleotides without dissociation from the template. Furthermore, we show that Mip1 can perform DNA synthesis on double-stranded templates utilizing a strand displacement mechanism. Our observations confirm that in contrast to its homologues in other organisms, Mip1 can function as a single-subunit replicative polymerase.

High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli.

We have developed a new expression vector, pcI(ts) ind(+), based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector "portable" in that it can be used to transform any strain of Escherichia coli. Hence, control over strains, induction conditions, and harvest times can be used to optimize yields of heterologous proteins. To provide a proof of concept, we show that E. coli recA(+) and recA(-) host cells transformed with pcI(ts) ind(+) modKlenTaq1 (a modified version of the large fragment of Thermus aquaticus DNA polymerase I) could be grown to high cell densities in multiple shake-flasks. A mutant version of modKlenTaq1 (V649C) could be induced by simply raising the thermostat setting from 30 to 37 degrees C and (in the case of recA(+) cells) adding nalidixic acid to achieve full induction (12-13% of the total cellular protein). Using a rapid, two-step purification process, it was possible to purify nearly 300 mg of modKlenTaq1 V649C from six 2.8-L baffle-bottomed shake-flasks each holding 1.5L of culture for a final yield of approximately 33 mg per liter or 3mg of purified enzyme per gram of cells wet weight.

NtPolI-like1 and NtPolI-like2, bacterial DNA polymerase I homologs isolated from BY-2 cultured tobacco cells, encode DNA polymerases engaged in DNA replication in both plastids and mitochondria.

Two cDNAs encoding homologs of bacterial DNA polymerase I were isolated from cultured tobacco (Nicotiana tabacum) BY-2 cells, and the corresponding genes were named NtPolI-like1 and NtPolI-like2. High sequence similarity suggested that they are orthologous genes each derived from respective parental species of N. tabacum, an allotetraploid plant. Each of the NtPolI-like1/2 gene products had a putative transit peptide for plastid localization at the N-terminus, followed by a 3'-5' exonuclease domain in the internal region, and a DNA polymerase domain in the C-terminal region. Among family A DNA polymerases, NtPolI-like proteins formed, together with other plant DNA polymerase I homologs, a phylogenetic group distinct from mitochondrial DNA polymerase gamma in animals and fungi, as well as eukaryotic cell nuclear-localized repair enzymes. In contrast to computer predictions, experiments with green fluorescent protein (GFP) fusion protein and Western blotting analysis suggested dual targeting of the gene products to both plastids and mitochondria. The recombinant NtPolI-like2 protein exhibited DNA polymerase activity in vitro. Their biochemical character roughly coincided with those of the 116 kDa DNA polymerases found in the plastid and mitochondrial nuclei (nucleoids) isolated from BY-2 cells. Pre-treatment of the organelle nuclear extracts with anti-NtPolI-like antibody removed most of the DNA polymerase activity. Reverse transcription-PCR (RT-PCR) and Western blotting analyses demonstrated transient activation of NtPolI-like gene expression in the initial phase of cell proliferation, exactly when the 116 kDa DNA polymerases in the isolated organelle nuclei were activated and preferential synthesis of organelle DNAs occurred. Taken together, our results suggest that NtPolI-like1/2 genes encode DNA polymerases engaged in DNA replication in both plastids and mitochondria.

Preliminary characterization of a thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41.

A thermostable DNA polymerase I from a mesophilic Bacillus sphaericus strain C3-41 was characterized in this study. The polI was cloned, sequenced and over-expressed in Escherichia coli. The expressed 110 kDa fusion protein of PolI was stable at 70 degrees C for 1 h. Compared with DNA polymerase I of E. coli (TaKaRa), the relative polymerase activity of this PolI was 3.33 +/- 0.1 RFU microl(-1) at 37 degrees C using fluorescent quantitative analysis. It showed higher polymerase activity than E. coli PolI at higher temperature, with a relative activity of 3.75 +/- 0.1 RFU microl(-1) at 70 degrees C. The polI sequence analysis and the protein structure prediction indicated that this protein had a high similarly to other PolI from thermophilic micro-organisms. This information is of importance for future study for evolution of the house-keeping gene polI in entomopathogenic bacterium B. sphaericus.

Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes One-step purification of Pfu DNA polymerase.

The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (K(D)) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.

Isolation and characterization of a DNA synthesome from a neuroblastoma cell line.

BACKGROUND: Despite significant analysis of the chromosomal abnormalities associated with neuroblastoma (NB), the role that NB DNA replication may play in the accumulation of genetic damage is poorly understood. For that matter, the mechanisms involved in NB DNA synthesis have yet to be elucidated. In an effort to investigate this process in NB, we have isolated and purified a multiprotein DNA replication complex from human NB cells (IMR-32). METHODS: Using a series of subcellular fractionations, ion-exchange chromatography, and gradient sedimentation steps, we have isolated a simian virus 40 replication competent multiprotein complex from IMR-32 NB cells, which has been designated the DNA synthesome. Enzymatic and immunodetection techniques were used to characterize the multiple components of the multiprotein DNA replication complex. RESULTS: The NB DNA synthesome was found to remain intact and functional through all the steps of its purification. The proteins and enzymatic activities that were found to copurify with the NB DNA synthesome include: DNA polymerases alpha , delta , and epsilon , proliferating cell nuclear antigen, replication factor A, replication factor C, topoisomerases I and II, flap endonuclease 1, and DNA ligase I. CONCLUSION: Although the cooperative integration of a DNA replication macromolecular complex (DNA synthesome) is not new, we extend the view of the DNA synthesome mediating DNA synthesis for human NB. The data reported here characterize the human NB DNA synthesome for the first time and provide the groundwork for investigating whether the NB DNA synthesome contributes to faulty DNA replication and tumor pathogenesis for this childhood malignancy.

Presence of 18-A long hydrogen bond track in the active site of Escherichia coli DNA polymerase I (Klenow fragment). Its requirement in the stabilization of enzyme-template-primer complex.

The analysis of the active site region in the crystal structures of template-primer-bound KlenTaq (Klenow fragment equivalent of Thermus aquaticus polymerase I) shows the presence of an approximately 18-A long H-bonding track contributed by the Klenow fragment equivalent of Asn(845), Gln(849), Arg(668), His(881), and Gln(677). Its location is nearly diagonal to the helical axis of the template-primer. Four base pairs in the double stranded region proximal to 3' OH end of the primer terminus appear to interact with individual amino acid components of the track through either the bases or sugar moieties. To understand the functional significance of this H-bonding network in the catalytic function of Klenow fragment (KF), we generated N845A, N845Q, Q849A, Q849N, R668A, H881A, H881V, Q677A, and Q677N mutant species by site-directed mutagenesis. All of the mutant enzymes showed low catalytic activity. The kinetic analysis of mutant enzymes indicated that K(m)(.dNTP) was not significantly altered, but K(D)(.DNA) was significantly increased. Thus the mutant enzymes of the H-bonding track residues had decreased affinity for template-primer, although the extent of decrease was variable. Most interestingly, even the reduced binding of TP by the mutant enzymes occurs in the nonproductive mode. These results demonstrate that an H-bonding track is necessary for the binding of template-primer in the catalytically competent orientation in the pol I family of enzymes. The examination of the interactive environment of individual residues of this track further clarifies the mode of cooperation in various functional domains of pol I.

Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner.

To achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography, a novel basic protein domain is used as a purification handle. The proteolytic instability usually encountered for basic peptide tags is avoided by the use of a highly constrained alpha-helical domain based on staphylococcal protein A into which positively charged amino acids have been introduced. Here we show that this domain, consisting of 58 amino acids with a calculated isoelectric point (pI) of 10.5, can be used to efficiently capture different fused target proteins, such as a bacterial DNA polymerase (Klenow fragment), a viral protease (3C) and a fungal lipase (Cutinase). In contrast to standard cation-exchange chromatography, efficient capture can be achieved also at a pH value higher than the pI of the fusion protein, demonstrated here by Zbasic-Klenow polymerase (pI approximately/= 5.8) and ZZ-Cutinase-Zbasic (pI approximately/= 7.2) both purified at a pH of 7.5. These results show that the Zbasic domain is able to confer a regional concentration of positive charge on the fusion protein even at a relatively high pH. Hence, the data suggest that this domain could be used for highly efficient and selective capture of target proteins at conditions where most host-cell proteins do not bind to the chromatographic resin. The obtained purity after this one-step procedure suggests that the strategy could be an alternative to standard affinity chromatography. Methods for site-specific proteolysis of the fusion proteins to release native target proteins are also discussed.

Biotinylated lithocholic acids for affinity chromatography of mammalian DNA polymerases alpha and beta.

Biotinylated lithocholic acids have been synthesized. The compounds inhibited mammalian DNA polymerases alpha and beta with dose-dependent manner. The streptavidine columns conjugated with the synthetic biotinylated compounds chromatographed both two enzymes eluted by KCl solution at the different concentrations.

A mutation in subunit B of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells concomitant with a conformational change and abnormal catalytic properties of the DNA polymerase alpha-primase complex.

Mutated constituents of the DNA replication complex might contribute to the mutational load of the genome during tumor development by impairing DNA synthesis as well as cell cycle-related control of DNA replication. To prove or disprove this hypothesis, we looked for mutations in the cDNA sequences of the four subunits of DNA polymerase alpha-primase from both highly malignant Novikoff hepatoma cells and regenerating normal rat liver and compared physicochemical and catalytic properties of the DNA polymerase alpha-primase complexes purified from both sources. Sequence analysis showed two mutations in subunit B from Novikoff cells: one in nucleotide position 855 (CCG-->CCA) that did not result in an amino acid exchange and one in position 862 (GTG-->ATG) that caused a change of valine to methionine in codon 288. No mutation was found in the three other subunits. The wild-type and mutated sequences of subunit B were cloned and expressed in vitro. Sedimentation analysis of the expressed polypeptides revealed different sedimentation constants, indicating that the amino acid exchange affected the conformation of subunit B. The analysis of the purified DNA polymerase alpha-primase complexes showed a sedimentation value that was significantly higher for the enzyme complex from normal liver than for that from Novikoff cells. In addition, DNA polymerase alpha-primase complexes from Novikoff cells showed higher sensitivity to camptothecin, topotecan, and structurally related compounds (such as (R,S)-7-ethyl-10-hydroxy camptothecin, 9-aminocamptothecin, and 10-hydroxycamptothecin) than the enzyme from normal rat liver. Thus, the amino acid change found in subunit B appears to result in a conformational change of the DNA polymerase alpha-primase complex from Novikoff hepatoma cells. Whether this mutation influences genetic instability or tumor development needs to be explored.

Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis.

Previous reports suggest that Treponema pallidum bacteremia occurs in persons with syphilis exposure ('incubating syphilis') and in persons with primary or secondary syphilis. During a recent syphilis outbreak, whole blood samples from 32 persons with suspected syphilis or syphilis exposure were screened using polymerase chain reaction (PCR) to amplify the DNA polymerase I gene (polA) of T. pallidum. Of the 32 samples, polA was amplified from 13 (41%). Of these 13, three were determined to have incubating syphilis; two had primary or secondary syphilis and eight had latent syphilis. This study demonstrates that spirochetemia can occur throughout the course of T. pallidum infection.

Amino acids 257 to 288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase alpha-primase to synthesize DNA in vitro.

Although p48 is the most conserved subunit of mammalian DNA polymerase alpha-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase alpha is controlled by E2F, an Ets-related transcription factor, and Sp1.

We have isolated a genomic DNA fragment spanning the 5'-end of the gene encoding the catalytic subunit of mouse DNA polymerase alpha. The nucleotide sequence of the upstream region was G/C-rich and lacked a TATA box. Transient expression assays in cycling NIH 3T3 cells demonstrated that the GC box of 20 bp (at nucleotides -112/-93 with respect to the transcription initiation site) and the palindromic sequence of 14 bp (at nucleotides -71/-58) were essential for basal promoter activity. Electrophoretic mobility shift assays showed that Sp1 binds to the GC box. We also purified a protein capable of binding to the palindrome and identified it as GA-binding protein (GABP), an Ets- and Notch-related transcription factor. Transient expression assays in synchronized NIH 3T3 cells revealed that three variant E2F sites near the transcription initiation site (at nucleotides -23/-16, -1/+7 and +17/+29) had no basal promoter activity by themselves, but were essential for growth-dependent stimulation of the gene expression. These data indicate that E2F, GABP and Sp1 regulate the gene expression of this principal replication enzyme.

Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans.

The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli. As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases. The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E. coli strain BL21(DE3)pLysS using the pARHS2 expression vector. The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure. The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Catalytic chromatography.

Catalytic chromatography exploits both specific biological affinity and catalytic specificity to selectively purify enzymes. Two different applications are presented. Purification of EcoRI restriction endonuclease to apparent homogeneity was accomplished in a single step with significantly greater yield and purification than was obtained with affinity chromatography. An attempt to purify the multiple DNA polymerase activities of Escherichia coli was also developed. Five well-resolved peaks of DNA polymerase activity were fractionated. In this new chromatographic mode, the enzyme binds immobilized substrate coupled to a column in the absence of some required cofactor. When the missing cofactor is added, the enzyme converts substrate to product and selectively elutes from the column.