Nucleotide Polymerization Research Articles

Biochemical characterization of a hepatitis C virus RNA-dependent RNA polymerase mutant lacking the C-terminal hydrophobic sequence.

The RNA-dependent RNA polymerase activity of hepatitis C virus is carried out by the NS5B protein. The full-length protein was previously purified as a non-fusion protein from insect cells infected with a recombinant baculovirus. The characterization is now described of a C-terminal hydrophobic domain deletion mutant of NS5B purified from E. coli. In addition to increased solubility, deletion of this sequence also positively affected the polymerase enzymatic activity. The efficiency of nucleotide polymerization of both the full-length and the C-terminal truncated enzymes were compared on homopolymeric template-primer couples as well as on RNA templates with heteropolymeric sequences. The largest difference in the polymerase activity was observed on the latter. On all the templates, the increased activity could be ascribed, at least in part, to enhanced template turnover of the deletion mutant with respect to the full-length enzyme. The elongation rates of the two enzyme forms were compared under single processive cycle conditions. Under these conditions, both the full-length and the deletion mutant were able to incorporate about 700 nt/min.

DNA polymerase beta-like nucleotidyltransferase superfamily: identification of three new families, classification and evolutionary history.

A detailed analysis of the polbeta superfamily of nucleotidyltransferases was performed using computer methods for iterative database search, multiple alignment, motif analysis and structural modeling. Three previously uncharacterized families of predicted nucleotidyltransferases are described. One of these new families includes small proteins found in all archaea and some bacteria that appear to consist of the minimal nucleotidyltransferase domain and may resemble the ancestral state of this superfamily. Another new family that is specifically related to eukaryotic polyA polymerases is typified by yeast Trf4p and Trf5p proteins that are involved in chromatin remodeling. The TRF family is represented by multiple members in all eukaryotes and may be involved in yet unknown nucleotide polymerization reactions required for maintenance of chromatin structure. Another new family of bacterial and archaeal nucleotidyltransferases is predicted to function in signal transduction since, in addition to the nucleotidyltransferase domain, these proteins contain ligand-binding domains. It is further shown that the catalytic domain of gamma proteobacterial adenylyl cyclases is homologous to the polbeta superfamily nucleotidyltransferases which emphasizes the general trend for the origin of signal-transducing enzymes from those involved in replication, repair and RNA processing. Classification of the polbeta superfamily into distinct families and examination of their phyletic distribution suggests that the evolution of this type of nucleotidyltransferases may have included bursts of rapid divergence linked to the emergence of new functions as well as a number of horizontal gene transfer events.

Enhanced impairment of chain elongation by inhibitors of HIV reverse transcriptase in cell-free reactions yielding longer DNA products.

We have studied the relationship between the length of HIV-1 reverse transcriptase (RT)-mediated nucleotide polymerization and inhibitors of these reactions in cell-free RT assays performed in the presence of either of two dideoxynucleoside triphosphates (ddNTPs), i.e. AZTTP or 3TCTP, or nevirapine, a non-nucleoside RT inhibitor. These reactions employed a heterologous RNA template and three DNA oligonucleotide primers, i.e. pAR, dPR and PA, that yielded distinct full-length products of 65, 192 and 376 nt, respectively, in the absence of inhibitor. We now show that the extent of inhibition of RT activity was greatest with use of the PA primer, which normally yielded the longest reaction product, and that lesser degrees of inhibition were noted in the reactions that generated shorter products. For example, at a concentration of 5 microM AZTTP, the extent of inhibition was 75% with the PA primer but only 40% and <10% when reactions were primed by the dPR and pAR primers, respectively. Similar results were obtained when either a mutated form of HIV RT (i.e. M184V), associated with resistance to 3TC, was tested in the presence of 3TCTP or when RT derived from Moloney murine leukemia virus (M-MuLV) was tested in the presence of AZTTP.

Toprim--a conserved catalytic domain in type IA and II topoisomerases, DnaG-type primases, OLD family nucleases and RecR proteins.

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of approximately 100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact beta/alpha fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by site-directed mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg2+that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.

Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

Amino Acid Changes in a Unique Sequence of Bacteriophage T7 DNA Polymerase Alter the Processivity of Nucleotide Polymerization

T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction. We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Delta5) to grow on E. coli having reduced thioredoxin levels. One triple mutant (K300E,K302E,K304E) could not support the growth of T7Delta5 even in wild type cells. This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin. The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex. The altered polymerase also has a lower binding affinity for double-stranded DNA.

Structural and Functional Organization of the DNA Polymerase of Bacteriophage T7

The 80-kDa gene 5 protein encoded by bacteriophage T7 shares significant amino acid homology with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment). Like the Klenow fragment, T7 gene 5 protein has both DNA polymerase and 3' to 5' exonuclease activities. However, unlike the Klenow fragment, T7 gene 5 protein binds tightly to E. coli thioredoxin to form a complex that has a high processivity of nucleotide polymerization. In order to identify the domains of gene 5 protein responsible for polymerization, hydrolysis, and binding of thioredoxin, we have analyzed proteolytic fragments of gene 5 protein. Cleavage of the protein within one protease-sensitive region (residue 250-300) yields two molecular weight species of peptides of 32-37 and 43-51 kDa derived from the N-terminal and C-terminal region, respectively. DNA polymerase activity is found within the C-terminal fragments and exonuclease activity within the N-terminal fragments. Thioredoxin stimulates the DNA polymerase activity of the C-terminal fragments. All fragments bind to DNA. In addition to delineating the polymerase and exonuclease domains, the protease-sensitive region appears to interact with E. coli thioredoxin. Thioredoxin protects this region from proteolysis, and alteration of this region reduces the ability of thioredoxin to stimulate polymerase activity.

Stimulation of Drosophila Mitochondrial DNA Polymerase by Single-stranded DNA-binding Protein

Mitochondrial DNA polymerase from Drosophila embryos has been characterized with regard to its mechanism of DNA synthesis in the presence of single-stranded DNA-binding protein from Escherichia coli. The rate of DNA synthesis by DNA polymerase gamma was increased nearly 40-fold upon addition of single-stranded DNA-binding protein. Processivity of mitochondrial DNA polymerase was increased approximately 2-fold, while its intrinsic rate of nucleotide polymerization was unaffected. Primer extension analysis showed that the rate of initiation of DNA strand synthesis by DNA polymerase gamma was increased 25-fold in the presence of single-stranded DNA-binding protein. Our results indicate that the stimulation of Drosophila DNA polymerase gamma by single-stranded DNA-binding protein results primarily from an increased rate of primer recognition and binding. Concurrent achievement of maximal activity and processivity by mitochondrial DNA polymerase in the presence of binding protein suggests that DNA polymerase gamma, like other replicative DNA polymerases, associates with accessory factors in vivo to catalyze efficient and processive DNA synthesis.

High-performance liquid chromatographic analysis of low-molecular-mass products synthesized by polynucleotide phosphorylase in polymerization reaction

HPLC was used for the simultaneous analysis of low- and high-molecular-mass products of nucleotide polymerization. Oligonucleotides with chain lengths of less than 5 monomer units were found in the reaction mixture at the very beginning of the reaction. These oligomers were not the products of non-specific polymer degradation. An increase in NaCl concentration from 0 to 1 M resulted in the appearance of a short lag period and the oligonucleotide accumulation. A simultaneous increase in Mg 2+ concentration eliminated the lag. It is concluded that high ionic strength affects both the formation of the metal-monomer-polymer complex and the affinity of the polynucleotide phosphorylase molecule for the reaction products.

Radionuclide-induced evolution of DNA and the origin of life.

Artesian groundwaters of high radionuclide concentration are ubiquitous and may have provided the large, sustained energy sources that were required to drive the multistage process of DNA and primordial cell evolution. The rapid, early development of the genetic code as well as its degeneracy can be attributed to exceptionally high radiation-induced mutation rates in this unique environment. The ability of double-strand DNA to direct enzymatic repair of radiation damage to single strands contributed importantly to its selective evolution. It is postulated that the polymerization of nucleotides took place at elevated temperatures within alpha-particle tracks of high ion and free-radical density, followed by rapid quenching to ambient conditions. It also is evident that radiation resistance and ploidy were important selection factors in cellular evolution.

Mitochondrial DNA polymerase from Drosophila melanogaster embryos: kinetics, processivity, and fidelity of DNA polymerization.

The mitochondrial DNA polymerase from embryos of Drosophila melanogaster has been examined with regard to template-primer utilization, processivity, and fidelity of nucleotide polymerization. The enzyme replicates predominantly single-stranded and double-stranded DNAs: the rate of DNA synthesis is greatest on the gapped homopolymeric template poly(dA).oligo(dT), while the highest substrate specificity is observed on single-stranded DNA templates of natural DNA sequence. Kinetic experiments and direct physical analysis of DNA synthetic products indicate that the Drosophila DNA polymerase gamma polymerizes nucleotides by a quasi-processive mechanism. The mitochondrial enzyme demonstrates a high degree of accuracy in nucleotide incorporation which is nearly identical with that of the replicative DNA polymerase alpha from Drosophila embryos. Thus, the catalytic properties of the near-homogeneous Drosophila DNA polymerase gamma are consistent with the in vivo requirements for mitochondrial DNA synthesis as described in a variety of animal systems.

Search for catalytic properties of simple polypeptides.

Simple polypeptides were used as possible supports for nucleotide polymerization, in the absence of any preformed polynucleotide template. Sequential copolymers of alanine and glycine, water soluble polypeptides based on arginine and poly(Glu-Ser-Glu) have been tested. No catalytic effect has been found although poly(Glu-Ser-Glu) favors the 2'-5' internucleotide linkage. More interestingly, polypeptides containing arginine residues strongly accelerate the hydrolysis of oligoadenylic acids. The influence of pH, temperature, nature of the buffer and polypeptide sequence was investigated.

Structure of large fragment of Escherichia coli DNA polymerase I complexed with dTMP.

The 3.3-A resolution crystal structure of the large proteolytic fragment of Escherichia coli DNA polymerase I complexed with deoxythymidine monophosphate consists of two domains, the smaller of which binds zinc-deoxythymidine monophosphate. The most striking feature of the larger domain is a deep crevice of the appropriate size and shape for binding double-stranded B-DNA. A flexible subdomain may allow the enzyme to surround completely the DNA substrate, thereby allowing processive nucleotide polymerization without enzyme dissociation.

A derivative of rifamycin SV inhibiting rifampicin-resistant RNA polymerase of Escherichia coli

The antibiotic rifampicin, a semi-synthetic derivative of rifamycin SV, inhibits bacterial RNA polymerase by forming a very stable complex with the enzyme in a 1: 1 ratio [ 11. In the search for rifamycins which would affect rifampicin-resistant bacteria, a large number of derivatives with bulky lipophilic side-chains were synthesized. Many of them proved capable of inhibiting the RNA polymerase of rifampicin-resistant E.coli mutants [2] and a number of other enzymes responsible for the polymerization of nucleotides [3]. However, the mechanism of action of these compounds against rifampicin-resistant RNA polymerases is different from the mechanism whereby rifampicin acts on the normal RNA polymerase. For example, the beststudied of these rifamycin derivatives, AF/013, unlike rifampicin prevents the binding of RNA polymerase to DNA [2,4]. The first rifamycin derivatives inhibiting a rifampicin-resistant Escherichia coli RNA polymerase by a similar mechanism to the action of rifampicin on a sensitive RNA polymerase were found among the dimeric rifamycins [5]. We have synthesized a number of derivatives of rifamycin SV with various substitutions in the 3rd position of the naphthoquinone nucleus; some of them proved capable of inhibiting rifampicin-resistant RNA polymerase of E.coli mutants. This paper describes a derivative of rifamycin SV, 3-(6-bromo-4-phenyl-2-hydrazonomethyl-quinazoline)-rifamycin SV (fig. 1) which Antibiotic resistance RM41 DNA-binding

Replication signals in prokaryotic DNA.

Synthesis of daughter strand DNA occurs by an ordered process of nucleotide polymerization which is under the control of the complementary sequence in the template parental strand. DNA duplication is catalyzed by DNA polymerase III in cooperation with a number of associated replication proteins; the DNA elongation complex has been termed “replisome” in the expectation of an ordered structure carrying out an ordered sequence of reaction steps.

On the role of the dnaB protein of Escherichia coli in the replication of lambda bacteriophage DNA.

The interaction between the dnaB protein of E. coli and the gene P product of lambda bacteriophage was investigated by measuring the cleavage of closed circular phage DNA after infection of two temperature sensitive dnaB mutants, JG28 and To534 groP- B. Cleavage of superhelical DNA from a lambda pi B mutant phage was observed after infection of either strain whereas superhelical DNA from a wild type phage was only cleaved after infection of JG28. When DNA synthesis in infected cells was blocked by incubation at the nonpermissive temperature, no inhibition of superhelical phage DNA cleavage was observed. It is concluded that in conditions where the dnaB protein has lost the capacity to function in nucleotide polymerization, it is capable of interacting with the lambda replication gene products to introduce a break in the phage DNA.

The catalysis of nucleotide polymerization by compounds of divalent lead

Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5'-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.

Purification of a high molecular weight human terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyltransferase has been purified from lymphoblasts of leukemic patients. The enzyme has a molecular weight of approximately 62,000 as determined by gel filtration and nondenaturing gel electrophoresis and is not dissociated into subunits by sodium dodecyl sulfate. In contrast, the terminal transferase enzyme from calf thymus has a molecular weight of 42,000 as determined by gel filtration, and is dissociated into 2 subunits of Mr 30,000 and 8,000 by sodium dodecyl sulfate. The enzyme has an isoelectric point of 8.2 and kinetic characteristics which are similar to those of calf thymus terminal transferase. The apparent Km for purine nucleotide polymerization at saturating initiator concentration with Mg2+ is 0.2 mM and with Mn2+ is 0.05 mM. Like calf terminal transferase, the reaction velocity is higher in the presence of Mg2+ than Mn2+. ATP inhibits the reaction catalyzed by terminal transferase isolated from human lymphoblasts due to mutual recognition of ATP and dATP by a common site on the enzyme. Preliminary experiments indicate that human terminal transferase may contain a small amount of carbohydrate. This report represents the first purification to near homogeneity of terminal transferase from a tissue source other than calf thymus.

ACTION OF KI AND SEVERAL IODOCOMPOUNDS ON [3H]URIDINE INCORPORATION INTO THYROID RNA

The present studies were performed in order to further clarify the action of iodine and iodocompounds on the incorporation of labelled uridine into thyroid RNA. KI decreased RNA labelling but did not alter total [3H]uridine uptake or [3H]inulin distribution space. KI also inhibited the increase in RNA labelling produced by 8 mM glucose. T4 was more potent on a molar basis than KI in impairing uridine incorporation. TETRAC, TRIAC and isopropyl-T3 also decreased RNA labelling, while T2 and isopropyl-T2 were ineffective. KI did not alter the distribution of the uridine derivatives, UMP, UDP and UTP, as determined by the distribution of [3H]uridine in these compounds by paper chromatography, suggesting that the action of KI does not take place at the step of uridine phosphorylation. Like its effect on TSH, KI also impairs the stimulatory effect of exogenous cAMP and cGMP on RNA labelling, suggesting that its action is exerted beyond the step of cyclic nucleotide formation. Iodine and iodocompounds may exert their inhibitory action on RNA labelling at the step of nucleotide polymerization.