Cellular DNA Polymerases Research Articles

Crosstalk between Primase Subunits Can Act to Regulate Primer Synthesis in trans

The coordination of primase function within the replisome is an essential but poorly understood feature of lagging strand synthesis. By using crystallography and small-angle X-ray scattering (SAXS), we show that functional elements of bacterial primase transition between two dominant conformations: an extended form that uncouples a regulatory domain from its associated RNA polymerase core and a compact state that sequesters the regulatory region from the site of primer synthesis. FRET studies and priming assays reveal that the regulatory domain of one primase subunit productively associates with nucleic acid that is bound to the polymerase domain of a second protomer in trans. This intersubunit interaction allows primase to select initiation sites on template DNA and implicates the regulatory domain as a "molecular brake" that restricts primer length. Our data suggest that the replisome may cooperatively use multiple primases and this conformational switch to control initiation frequency, processivity, and ultimately, Okazaki fragment synthesis.

Understanding How the Herpes Thymidine Kinase Orchestrates Optimal Sugar and Nucleobase Conformations To Accommodate Its Substrate at the Active Site: A Chemical Approach

The herpes virus thymidine kinase (HSV-tk) is a critical enzyme for the activation of anti-HSV nucleosides. However, a successful therapeutic outcome depends not only on the activity of this enzyme but also on the ability of the compound(s) to interact effectively with cellular kinases and with the target viral or cellular DNA polymerases. Herein, we describe the synthesis and study of two nucleoside analogues built on a conformationally locked bicyclo[3.1.0]hexane template designed to investigate the conformational preferences of HSV-tk for the 2'-deoxyribose ring. Intimately associated with the conformation of the 2'-deoxyribose ring is the value of the C-N torsion angle chi, which positions the nucleobase into two different domains (syn or anti). The often-conflicting sugar and nucleobase conformational parameters were studied using North and South methanocarbadeoxythymidine analogues (6 and 7), which forced HSV-tk to make a clear choice in the conformation of the substrate. The results provide new insights into the mechanism of action of this enzyme, which cannot be gleaned from a static X-ray crystal structure.

Modifications of keratinocyte functions by human papillomaviruses

Modifications of keratinocyte functions by human papillomaviruses

Recent Advances in 4-Thionucleosides as Potential Antiviral and Antitumor Agents

The classical 4'-oxonucleoside analogs exhibit interesting biological activities such as antibiotic, antiviral and antitumor, which are believed to be the result of inhibition of the viral or cellular DNA or RNA polymerase after being converted to their corresponding 5'-triphosphates. However, the activity of 4'-oxonucleosides were limited by their susceptibility to degradation by nucleoside phosphorylases or acid hydrolysis. This aspect called for the chemical modification of the carbohydrate portion. This compulsion led to two kinds of strategies; (1) replacement of the 4'-oxygen by the methylene group - carbocyclic nucleosides; (2) replacement of the 4' oxygen by sulphur-4'-thionucleosides. This group has also conferred the resistance to the nucleoside cleavage. Although, there were some pioneering work on 4'-thionucleosides in 1960s and 1970s, the interest in this group of compounds was rekindled by the antiviral activities of 2'-deoxy-4'-thionucleosides reported independently by Secrist et al. and Walker et al. Subsequent contributions by the other authors, enhanced its standing as an important class of antiviral agents. Following is a reasonably exhaustive account of this class of compounds reported after 1990.

Novel Mechanism of Inhibition of HIV-1 Reverse Transcriptase by a New Non-nucleoside Analog, KM-1

2-Naphthalenesulfonic acid (4-hydroxy-7-[[[[5-hydroxy-6-[(4 cinnamylphenyl)azo]-7-sulfo-2-naphthalenyl]amino]-carbonyl]amino]-3-[(4-cinnamylphenyl)]azo (KM-1)) is a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) that was designed to bind at an unconventional site on human immunodeficiency virus type 1 reverse transcriptase (RT) (Skillman, A. G., Maurer, K. W., Roe, D. C., Stauber, M. J., Eargle, D., Ewing, T. J., Muscate, A., Davioud-Charvet, E., Medaglia, M. V., Fisher, R. J., Arnold, E., Gao, H. Q., Buckheit, R., Boyer, P. L., Hughes, S. H., Kuntz, I. D., and Kenyon, G. L. (2002) Bioorg. Chem. 30, 443-458). We have investigated the mechanism by which KM-1 inhibits wild-type human immunodeficiency virus type 1 RT by using pre-steady state kinetic methods to examine the effect of KM-1 on the parameters governing the single nucleotide incorporation catalyzed by RT. Analysis of the pre-steady-state burst phase of dATP incorporation showed that KM-1 decreased the amplitude of the reaction as previously shown for other NNRTIs, because of the slow equilibration of the inhibitor with RT. In the ternary enzyme-DNA-KM-1 complex (E-DNA-I), incorporation of the next nucleotide onto the primer is blocked. However, unlike conventional NNRTIs, the inhibitory effect was caused primarily by weakening the DNA binding affinity and displacing DNA from the enzyme. Wild-type RT binds a 25/45-mer DNA duplex with an apparent K(d) of 3 nm, which was increased to 400 nm upon saturation with KM-1. Likewise, the apparent K(d) for KM-1 binding to RT increased at higher DNA concentrations. We therefore conclude that KM-1 represents a new class of inhibitor distinct from nevirapine and related NNRTIs. KM-1 can bind to RT in both the absence and presence of DNA but weakens the affinity for DNA 140-fold so that it favors DNA dissociation. The data suggest that KM-1 distorts RT conformation and misaligns DNA at the active site.

Double Cyclization of Bis(α‐hetarylmethyl)amino Esters to Optically Active Bridged N‐Heterocycles of HIV‐Inhibiting Activity

AbstractAnellated 1‐azabicyclo[3.3.1]nonanes 6 were synthesized by several routes starting from natural α‐amino esters 2 and o‐haloaryl‐ or o‐bromohetarylmethyl bromides 1. N‐Alkylation of the starting amino esters to 5 and 3 was followed by halogen/lithium exchange and double cyclization. The cyclization products 6 exhibit interesting inhibition of RNase H and DNA‐polymerase activity of reverse transcriptase (RT) of HIV‐1 at concentrations where human cellular DNA polymerases are not affected. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Coordinated Regulation of Replication Protein A Activities by Its Subunits p14 and p32

The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPADeltap14, consisting of p70 and p32. RPADeltap14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADeltap14 complex p32 only minimally recognized the 3'-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPADeltap14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-primase, it did not support cell-free SV40 DNA replication. This inability manifested itself in a failure to support both the primer synthesis and primer elongation reactions. These data reveal that efficient binding and correct positioning of the RPA complex on single-stranded DNA requires all three subunits to support DNA replication.

84. Enhanced Detection Method for Retrovirus in Vaccine

Detecting any presence of retroviruses in biological material is important to ensure safety and prevent retroviral infection, which has been associated with various disorders including malignancies, immunodeficiencies, and neurological disorders. Highly specific and sensitive PCR techniques are used to detect specifically the presence of a known retrovirus. In contrast, the presence of an unknown retrovirus can only be examined by non-specific methods such as transmission electron microscopy and growth of virus on susceptible cells. However, detection methodologies involving cell culture techniques may be ineffective because certain retroviruses are not cytopathic in vitro. Retroviruses possess RNA-dependent DNA polymerase known as reverse transcriptase (RT), which is inherent in all kinds of retrovirus. Taking advantage of its ubiquitous nature in retroviruses, enzymatic activity of RT may be measured to detect retroviral contamination in biological products. On the basis of the polymerase enhanced reverse transcriptase (PERT) assay, two different assay systems, PCR-based RT assay (PERT-PCR) and ELISA-based RT assay (PERT-ELISA), were developed. Respective optimum conditions were determined. Sensitivity, linearity and reproducibility of the methods were studied on purified RT. Both PERT-PCR and PERT-ELISA could detect as little as 10-10 unit/ml of RT. Nonetheless, no additional increase in sensitivity was observed by performing ELISA after PCR amplification. Treatment with RNase after cDNA synthesis increased sensitivity over 100 times, and the addition of synthetic DNA competitors, which suppress RT-like activities of cellular DNA-dependent DNA polymerases, greatly increased specificity of the assay. A modified form of PERT-PCR assay was used and subsequently RT activities were detected in vaccines and culture supernatant of retrovirus-producing cells. This enhanced form of PERT-PCR assay is easy to perform and is able to detect up to 10-10 unit/ml of purified RT, as well as RT in vaccines and cell culture supernatant. Thus, this study presents an enhanced assay, applicable to screening of all replication-competent retrovirus, quantification of the viral load, drug susceptibility testing of RT, and control of virus inactivation in biological products.

A critique of the Montagnier evidence for the HIV/AIDS hypothesis

In 1983 Luc Montagnier and his colleagues claimed to have discovered a novel retrovirus presently known as human immunodeficiency virus (HIV). By 1984 HIV was almost universally accepted to be the cause of AIDS. However, 20 years later, HIV cannot account for the phenomena for which the retroviral hypothesis was proposed, namely, Kaposi's sarcoma, decrease in T4 lymphocytes and thus the opportunistic infections in AIDS patients which were assumed to be the direct results of this decrease. Agents other than HIV to which patients belonging to the AIDS risk groups are exposed cause decrease in T4 cells. Neither have the main predictions of the HIV hypothesis been fulfilled. HIV seropositivity in the developed countries still remains restricted to the original high risk groups, no HIV vaccine exists, and no successful animal model has been developed. In this communication, we critically analyse the evidence which in 1983 was claimed to prove the existence of HIV. The phenomena which Montagnier and his colleagues considered proof for the existence of HIV are detection of reverse transcriptase activity; the presence of retrovirus-like particles in the culture; immunological reactivity between proteins from the culture supernatant which, in sucrose density gradients, banded at the density of 1.16 g/ml (“purified virus”) and antibodies in a patient's (BRU) serum. Reverse transcriptase activity can be found in viruses other than retroviruses and in all normal cells. Reverse transcription can be brought about not only by the enzyme reverse transcriptase but also by normal, cellular DNA polymerases. Retrovirus-like particles are ubiquitous in cultures not infected with retroviruses, especially in conditions employed by Montagnier et al. From the reaction between proteins in the “purified virus” and antibodies in the patient serum Montagnier concluded that the proteins were HIV proteins and the antibodies were HIV antibodies. Since all antibodies are polyspecific, from such a reaction it is not possible to define the origin of even one reactant let alone both. Even if this were possible, since Montagnier's “purified virus” did not contain particles with the “morphology typical of retroviruses”, the proteins cannot be retroviral. We conclude that, these phenomena are non-specific to retroviruses and thus cannot be considered proof for the existence of a unique retrovirus HIV.

Differential alternative splicing expressions of telomerase reverse transcriptase in gastrointestinal cell lines

Telomerase is a cellular RNA-dependent DNA polymerase that serves to maintain the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends. One of the human telomerase components is hTERT, which has three alternative spliced sites that introduce eight isoforms of hTERT mRNA. The expression of these isoforms in gastrointestinal cell lines is unknown. We developed a PCR-based assay for detecting these splicing variants. In gastric and hepatocellular carcinoma cell lines, the gamma deletion variant and its combination variants, alpha- and gamma-, beta- and gamma-, and alpha-, beta- and gamma-deletion variants were frequently detected, while they were not detected in colorectal carcinoma cell lines. Our results provide important information of use for more detailed studies on the regulation of telomerase activity.

Experimental and structural evidence that herpes 1 kinase and cellular DNA polymerase(s) discriminate on the basis of sugar pucker.

Two isomers of methanocarba (MC) thymidine (T), one an effective antiherpes agent with the pseudosugar moiety locked in the North (N) hemisphere of the pseudorotational cycle (1a, N-MCT) and the other an inactive isomer locked in the antipodean South (S) conformation (1b, S-MCT) were used to determine whether kinases and polymerases discriminate between their substrates on the basis of sugar conformation. A combined solid-state and solution conformational analysis of both compounds, coupled with the direct measurement of mono-, di-, and triphosphate levels in control cells, cells infected with the Herpes simplex virus, or cells transfected with the corresponding viral kinase gene (HSV-tk), suggests that kinases prefer substrates that adopt the S sugar conformation. On the other hand, the cellular DNA polymerase(s) of a murine tumor cell line transfected with HSV-tk incorporated almost exclusively the triphosphate of the locked N conformer (N-MCTTP), notwithstanding the presence of higher triphosphate levels of the S-conformer (S-MCTTP).

Discovery of DNA Polymerase

Discovery of DNA Polymerase

Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases

Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases α and β and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (Ki = 0.51μM and Ki = 0.53μM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby Km remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.

Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases.

Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases alpha and beta and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (K(i)=0.51 microM and K(i)=0.53 microM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby K(m) remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation.

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.

Molecular basis of fidelity of DNA synthesis and nucleotide specificity of retroviral reverse transcriptases

Reverse transcription involves the conversion of viral genomic RNAinto proviral double-stranded DNA that integrates into the host cell genome. Cellular DNA polymerases replicate the integrated viral DNA and RNA polymerase II transcribes the proviral DNA into RNA genomes that are packaged into virions. Although mutations can be introduced at any of these replication steps, reverse transcriptase (RT) errors play a major role in retroviral mutation. This review summarizes our current knowledge on fidelity of reverse transcriptases. Estimates of retroviral mutation rates or fidelity of retroviral RTs are discussed in the context of the different techniques used for this purpose (i.e., retroviral vectors replicated in culture, misinsertion and mispair extension fidelity assay, etc.). In vitro fidelity assays provide information on the RT's accuracy during the elongation reaction of DNA synthesis. In addition, other steps such as initiation of reverse transcription, or strand transfer, and factors including viral proteins such as Vpr [in the case of the human immunodeficiency virus type 1 (HIV-1)] have been shown to influence fidelity. A comprehensive description of the effect of amino acid substitutions on the fidelity of HIV-1 RT is presented. Published data point to certain dNTP-binding residues, as well as to various amino acids involved in interactions with the template or the primer strand, and to residues in the minor groove-binding track as major components of the fidelity center of retroviral RTs. Implications of these studies include the design of novel therapeutic strategies leading to virus extinction, by increasing the viral mutation rate beyond a tolerable threshold.

Specific detection of RT activity in culture supernantants of retrovirus-producing cells, using synthetic DNA as competitor in polymerase enhanced reverse transcriptase assay

The polymerase enhanced reverse transcriptase (PERT) assay is a highly sensitive assay for the detection of reverse transcriptase (RT) activity in culture supernatants of retrovirus-producing cells. However, some cellular DNA-dependent DNA polymerases exhibit RT-like activities in this assay. A synthetic DNA competitor which suppresses the RT-like activities of cellular DNA-dependent DNA polymerases was used in a modified PERT assay technique for specific detection of RT activity in culture supernatants of retrovirus-producing cells. We determined the optimum condition of the assay and evaluated its specificity. This improved PERT assay is easy to perform and is able to detect minute amounts of purified RT, as well as RT in crude cell lysates and concentrated culture supernatants.

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA.

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.

Nonintegrated circular forms of the HIV-1 provirus DNA in experimental HIV infection.

In this study, various proviral HIV-1 DNA forms were analyzed in a chronically infected culture of human monocytes. To elucidate the role of viral RT and cellular DNA polymerases in the formation of the circular forms, we used azidothymidine (AZT) as a specific terminator of viral RT. We previously developed a method for testing the circular proviral DNA forms by means of polymerase chain reaction (PCR) [5] and specific pairs of primers located at the 3' and 5' ends of the HIV-1 DNA (in the env and gag gene regions, respectively). The primers were so oriented that both one-LTR and two-LTR circular proviral DNAs could be determined simultaneously [5]. The total HIV-1 DNA concentration was measured to prepare diluted samples with an equal amount of proviral DNA, in which the relative content of circular nonintegrated forms of viral DNA was detected by means of PCR. The primers nov1‐nov2 were used. An additional amplification with primers V1‐V2 or nov3‐nov4 was performed in samples with low two-LTR circular DNA concentrations.

Simple and rapid preparation of gapped plasmid DNA for incorporation of oligomers containing specific DNA lesions.

The cytotoxicity, mutagenicity, and carcinogenicity of DNA base lesions are largely determined by the responses of cellular DNA repair proteins, DNA polymerases, and signaling pathways. Elucidation of these processes is thus of high biochemical interest. Such studies increasingly rely on DNA substrates containing specific lesions at defined locations. Although short synthetic DNA oligomers have frequently proved useful, circular plasmid substrates are preferable for much biochemical work, and essential for in vivo studies. However, the complexity of current approaches for preparing such substrates and limitations inherent in the procedures have posed problems. We present here a simple, highly versatile procedure for preparing gapped duplex plasmids, into which oligomers incorporating specific lesions can easily be inserted. Endonuclease N.BstNBI was used to nick twice the same strand of a pUC19-derived plasmid (pUC19HBDa), at two GAGTCNNNN sequences separated by 22 bases. Removal of the 22-nt oligomer and further purification produced a highly pure gapped plasmid. To illustrate application of this procedure, 22-nt oligonucleotides containing a single uracil residue were ligated into the gapped molecules. The pUC19HB(Da) plasmid can be modified to accept almost any DNA-lesion-containing oligomer. Using this new approach to incorporate specific DNA lesions into popular reporter genes will facilitate in vivo study of cellular responses to DNA damage.