Biogenesis of multisubunit RNA polymerases.

Gene transcription in the nucleus of eukaryotic cells is carried out by three related multisubunit RNA polymerases, Pol I, Pol II and Pol III. Although the structure and function of the polymerases have been studied extensively, little is known about their biogenesis and their transport from the cytoplasm (where the subunits are synthesized) to the nucleus. Recent studies have revealed polymerase assembly intermediates and putative assembly factors, as well as factors required for Pol II nuclear import. In this review, we integrate the available data into a model of Pol II biogenesis that provides a framework for future analysis of the biogenesis of all RNA polymerases.

The Pole3 bidirectional unit is regulated by MYC and E2Fs.

Pole3 (DPB4/YBL1/CHRAC17) is one of the subunits of the DNA polymerase e. It contains a histone-like domain required for the hererodimerization with its Pole4 (DPB3) partner. In another interaction, Pole3 heterodimerizes with YCL1/CHRAC15 and associates with the ACF1/SNF2H remodelling complex. We find that the Pol3 gene is regulated in starved NIH3T3 fibroblasts upon induction with serum, with a peak at the entry in the S phase. We characterized the Pole3 promoter, which is linked bidirectionally to C9Orf46, a gene of unknown function: it has no CCAAT nor TATA-boxes, and contains an E box and two potential E2F sites. Mutagenesis analysis points to a minimal promoter region as sufficient for activation; the E box and a neighbouring direct repeat are important for regulation. Cell-cycle regulation was reproduced in stable clones and an additional E2F site was found to be important. Chromatin immunoprecipitation analysis indicates that E2F1/4, as well as MYC, are associated with the Pole3 promoter in a phase-specific way. These data highlight coregulation of a histone-like gene with core histones upon DNA synthesis, and represent a first dissection of the interplay between two essential cell-cycle regulators on a bidirectional promoter.

Exploiting cis-acting replication elements to direct hepatitis C virus-dependent transgene expression.

We describe here a novel targeting gene therapy strategy to direct gene expression responsive to hepatitis C virus (HCV). The goal was approached by engineering a construct containing the antisense sequence of the transgene and internal ribosome entry site of encephalomyocarditis virus flanked by 5'- and 3'-end sequences of HCV cDNA that contain cis-acting replication elements. Thus, expression of the transgene is only promoted when the minus-strand RNA has been synthesized by the functional replication machinery present in infected cells. Reporter assay and strand-specific reverse transcription-PCR showed selective transgene expression in Huh-7 cells harboring an autonomously replicating HCV subgenome but remaining silent in uninfected cells. Furthermore, using the cytosine deaminase suicide gene as a transgene coupled with recombinant adenovirus delivery, we demonstrated that cytosine deaminase was specifically expressed in replicon cells, resulting in marked chemosensitization of replicon cells to the cytotoxic effects of flucytosine. This new targeting strategy could be extended to other single-stranded RNA viruses encoding the unique RNA-dependent RNA polymerase that has no parallel in mammalian cells.

Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations.

BACKGROUND: The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. METHODS: A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. RESULTS: Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2-4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed significant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA structure. CONCLUSIONS: This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

DNA polymerases and Ki-67 nuclear antigen are induced in correlation with the resected mass of rat liver up to 90%.

BACKGROUND AND AIMS: We studied the regeneration potential by measuring induction of DNA polymerases in the remnant rat liver after a partial hepatectomy (PHx) that is maximal but compatible with survival. METHODS: The regenerating rat liver was obtained after the 90% PHx. The induction of activities of DNA polymerase alpha, delta, and epsilon were measured after partial purification. The Ki-67 nuclear antigen was also detected histochemically. These parameters were compared with those after both 30% and 70% PHx. RESULTS: The 90% hepatectomy resulted in the strong inductions of DNA polymerase alpha, delta, and epsilon, at 48 h after operation, in association with increases in wet weight and total DNA in the remnant liver. The enzyme induction was much higher after 90% PHx than after 30% and 70% hepatectomy, in correlation with the resection volume. At 48 h after 90% hepatectomy, the Ki-67 positive cells increased up to 47.2% of hepatocytes in the remnant liver. CONCLUSION: The higher induction of replication enzymes by 90% hepatectomy reflects more cells entering mitogenic cell cycle, which supports the fast regeneration of the remnant liver. The number of proliferating hepatocytes is stringently controlled by an unknown mechanism sensing the mass of resected liver parenchyma.

Evolution of internal eliminated segments and scrambling in the micronuclear gene encoding DNA polymerase alpha in two Oxytricha species.

To learn about the evolution of internal eliminated segments (IESs) and gene scrambling in hypotrichous ciliates we determined the structure of the micronuclear (germline) gene encoding DNA polymerasealpha(DNA polalpha) in Oxytricha trifallax and compared it to the previously published structure of the germline DNA polalphagene in Oxytricha nova . The DNA polalphagene of O.trifallax contains 51 macronuclear-destined segments (MDSs) separated by 50 IESs, compared to 45 MDSs and 44 IESs in the O.nova gene. This means that IESs and MDSs have been gained and/or lost during evolutionary divergence of the two species. Most of the MDSs are highly scrambled in a similar non-random pattern in the two species. We present a model to explain how IESs, non-scrambled MDSs and scrambled MDSs may be added and/or eliminated during evolution. Corresponding IESs in the two species differ totally in sequence, and junctions between MDSs and IESs are shifted by 1-18 bp in O.trifallax compared to the O.nova gene. In both species a short region of the gene is distantly separated from the main part of the gene. Comparison of the gene in the two species shows that IESs and scrambling are highly malleable over evolutionary time.

Ligation of portal vein branch induces DNA polymerases alpha, delta, and epsilon in nonligated lobes.

Ligation of a portal vein branch supplying 70% of the rat liver causes compensatory hypertrophy of the nonligated hepatic lobes with concomitant atrophy of the ligated lobes. To elucidate the mechanism of this response, the induction of the replication enzymes DNA polymerases alpha, delta, epsilon, as well as proliferating cell nuclear antigen (PCNA), were investigated in nonligated lobes after portal branch ligation. The induction patterns were compared with the well studied liver regeneration after 70% partial hepatectomy. DNA polymerases alpha, delta, and epsilon in the liver were extracted with 5 mM KCl (low-salt extract), then with 600 mM KCl (high-salt extract). DNA polymerases alpha, delta, and epsilon in low-salt extract were partially separated on a hydroxyapatite column and quantified. All enzyme activities in the nonligated lobes started to increase within 24 hr and reached maximum levels by 48 hr after portal branch ligation. These patterns were quite similar to those obtained with the remnant liver after partial hepatectomy. In low-salt extract, DNA polymerase delta and epsilon were prominent, while, in high-salt extract, largely DNA polymerases alpha and some activity of epsilon were recovered. PCNA was also induced after both portal branch ligation and partial hepatectomy, reaching maximum levels at 48 hr. From the similar changes in DNA polymerases and PCNA, our data indicate that portal branch ligation induces hepatocyte proliferation in the nonligated lobes in a way similar to partial hepatectomy.

Prostate-specific antigen promoter driven gene therapy targeting DNA polymerase-alpha and topoisomerase II alpha in prostate cancer.

Attainment of cell type-specific cytotoxicity with minimal side effects is the ultimate goal of cancer therapy. By employing the prostate-specific antigen promoter (PSAP), we investigated (1) whether PSAP-driven antisense genetic constructs targeting DNA polymerase-alpha and topoisomerase II alpha (Top II alpha), designated PSAP-antipol and PSAP-antitop respectively, could induce death of prostate cancer cells, and (2) whether the cytotoxicity is restricted to cells of prostate origin. A PSAP-driven beta-galactosidase gene, PSAP-LacZ, was also used to estimate the expression of the PSAP-driven transcripts. Lipofection-mediated gene transfers were performed with these 3 constructs and a control plasmid, pCDNA3, in 3 human prostate cancer cell lines (LNCaP, DU-145, PC-3) and 5 other cell lines (Cos-1 [monkey kidney], HL-60 [human myeloid leukemia], Hep G2 [human hepatoma], NCI H460 [human lung cancer] and SW 480 [human colon cancer]). On transfection with PSAP-LacZ, LNCaP, DU-145, and PC-3 showed a 10.8, 1.8, and 1.6 fold increase in beta-galactosidase activity, respectively. The remaining 5 cell lines showed no changes after transfection. Corresponding to the levels of the induced beta-galactosidase activity, LNCaP showed the strongest growth inhibition by the antisense constructs: 36% by PSAP-antipol, 39% by PSAP-antitop and 80% by PSAP-antipol+PSAP-antitop. DU-145 and PC-3 had minimal growth inhibition with PSAP-antipol alone or PSAP-antitop alone. However, when cotransfected with PSAP-antipol and PSAP-antitop, DU-145 and PC-3 displayed 42% and 55% growth inhibition, respectively. In contrast, no cytotoxicity was observed in the remaining 5 cell lines when transfected with PSAP-antipol, PSAP-antitop or both. Therefore, PSAP-driven antisense gene therapy targeting DNA polymerase-alpha and Top II alpha inhibits the growth of human prostate cancer cells and the cytotoxic effect is restricted in cells of prostate origin.

DNA replication-related elements cooperate to enhance promoter activity of the drosophila DNA polymerase alpha 73-kDa subunit gene.

An analysis was carried out on the promoter region of the Drosophila DNA polymerase alpha 73-kDa subunit gene and the factor(s) activating the promoter. Transcription initiation sites were newly identified in the region downstream of the previously determined sites. Full promoter activity resided within the region from -285 to +129 base pairs with respect to the newly determined major site. Within this region, we found three sequences identical or similar to the DNA replication-related element (DRE), 5'-TATCGATA, which is known as a common promoter-activating element for the Drosophila DNA polymerase alpha 180-kDa subunit gene and the proliferating cell nuclear antigen gene. These sites were located at positions -77 to -70 (DREalpha-I), -44 to -37 (DREalpha-II), and +3 to +10 (DREalpha-III). Footprinting analysis using the recombinant DRE-binding factor (DREF) or Kc cell nuclear extract demonstrated that DREF can bind to all three DRE-related sites. Introduction of mutation in even one of the three DRE-related sequences caused extensive reductions of the promoter activity and also the DREF-binding activity of the promoter-containing fragment. The results indicate that the three DREF-binding sites cooperate to enhance promoter activity of the DNA polymerase alpha 73-kDa subunit gene.

Inhibition of DNA polymerase alpha expression and cell growth, a possible triple helix mechanism.

The antiproliferative effects mediated by a 14-mer homopyrimidine oligonucleotide (5' CTTTCT-CTTTTCTC3'), designed to form DNA triplex with a purine region of the DNA polymerase alpha promoter, were evaluated on the human breast cancer cell line MDA-MB 231. In order to stabilize the triple complex under physiologic conditions, replacement of cytosines by methylcytosines in the oligomer sequence was carried out. Band-shift analyses demonstrated a complete triplex formation between the radiolabeled target duplex DNA and the methylcytosine-modified oligomer at the concentration of 0.1 microM under physiologic pH and temperature. A single exposure of MDA-MB 231 cells to 0.5 microM methylcytosine-modified oligonucleotide was able to markedly reduce the cell number and the percentage of cells in DNA synthesis up to 58% and 66%, respectively, compared with controls. Furthermore, a 48% reduction in the amount of the DNA polymerase alpha mRNA was reported after treatment with the oligomer. In conclusion, data from the present study demonstrate that an oligonucleotide to DNA polymerase alpha promoter, designed to form a triple helix with target double-stranded DNA, inhibits the expression of the reporter gene at the biologic and molecular levels, suggesting a possible triplex-mediated mechanism of action.

Cloning and complete sequence of the DNA polymerase-encoding gene (BstpolI) and characterisation of the Klenow-like fragment from Bacillus stearothermophilus.

A fragment of the DNA polymerase I-encoding gene (polI) from Bacillus stearothermophilus (Bst) was obtained by PCR. This was used as a probe to obtain a full-length gene from a Bst genomic DNA (gDNA) plasmid library. Comparison of the sequence to B. caldotenax (Bca) showed about 93% homology at the amino acid (aa) level. A Klenow-like (BstpolIk) clone was developed and the recombinant protein displayed DNA polymerase activity similar to the wild-type BstPolI enzyme.

Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes.

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.

Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha.

The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.

Purification and properties of wild-type and exonuclease-deficient DNA polymerase II from Escherichia coli.

Wild-type DNA polymerase II (pol II) and an exonuclease-deficient pol II mutant (D155A/E157A) have been overexpressed and purified in high yield from Escherichia coli. Wild-type pol II exhibits a high proofreading 3'-exonuclease to polymerase ratio, similar in magnitude to that observed for bacteriophage T4 DNA polymerase. While copying a 250-nucleotide region of the lacZ alpha gene, the fidelity of wild-type pol II is high, with error rates for single-base substitution and frameshift errors being < or = 10(-6). In contrast, the pol II exonuclease-deficient mutant generated a variety of base substitution and single base frameshift errors, as well as deletions between both perfect and imperfect directly repeated sequences separated by a few to hundreds of nucleotides. Error rates for the pol II exonuclease-deficient mutant were from > or = 13- to > or = 240-fold higher than for wild-type pol II, depending on the type of error considered. These data suggest that from 90 to > 99% of base substitutions, frameshifts, and large deletions are efficiently proofread by the enzyme. The results of these experiments together with recent in vivo studies suggest an important role for pol II in the fidelity of DNA synthesis in cells.

The efficiency of translesion synthesis past single styrene oxide DNA adducts in vitro is polymerase-specific.

In order to examine the effect of adenine N6 adducts of styrene oxide (SO) on DNA replication, 33-mer templates were constructed bearing site-specific and stereospecific SO modifications. Both R- and S-SO adducts were introduced at four different base positions within a sequence containing codons 60-62 from the human N-ras gene. The resulting eight templates were replicated in primer extension assays using the Klenow fragment, Sequenase 2.0, T4 polymerase holoenzyme, polymerase alpha, and polymerase beta. Replication of the damaged templates was analyzed under conditions defining single and/or multiple encounters between the polymerase and the substrate. Polymerization by all five enzymes was sensitive to both the local sequence context and the chirality of the SO adduct. For example, R-SO lesions placed at the third position of N-ras codon 61 were readily bypassed, whereas stereochemically-identical lesions in other sequence contexts were often poor substrates for replication. Similarly, R- and S-SO adducts introduced within identical sequences were often bypassed nonequivalently. Significantly, the degree of adduct-directed termination and translesion synthesis during replication was also dependent on the choice of polymerase. Although SO adducts directed termination either opposite the lesion or 1 base 3' to the damage using all five polymerases, templates that were poor substrates for bypass synthesis with one enzyme were often read-through much more efficiently when a different polymerase was used. Thus, the activities of these enzymes on the SO-modified substrates produced replication profiles, or "fingerprints", that were unique to each polymerase.

Expression patterns of DNA replication enzymes and the regulatory factor DREF during Drosophila development analyzed with specific antibodies.

Specific antibodies were prepared against Drosophila DNA polymerase epsilon and DREF, a regulatory factor for DNA replication-related genes. Using these antibodies together with those for DNA polymerase alpha and proliferating cell nuclear antigen (PCNA), we examined expression patterns and sub-cellular distributions of these proteins during Drosophila development. DNA polymerase alpha, epsilon and PCNA proteins were maternally stored in unfertilized eggs and maintained at high levels during embryogenesis. With distinct nuclear localization, proteins were observed in embryos at interphase stages throughout the 13 nuclear division cycles, suggesting that they all participate in rapid nuclear DNA replication during these cycles. In contrast, maternal storage of a DREF protein was relatively low and its level increased throughout embryogenesis. Strong nuclear staining with the anti-DREF antibody was not observed until the nuclear division cycle 8. Immunostaining of various larval tissues from transgenic flies carrying the PCNA gene promoter-lacZ fusion gene revealed co-expression of DREF, PCNA and lacZ, suggesting that DREF regulates the expression of PCNA gene in these tissues. In addition, we detected a relatively high level of DREF in adult males as well as females. Since DNA polymerase alpha, epsilon and PCNA are hardly detectable in adult males, DREF very likely regulates genes other than those closely linked to DNA replication in adult males.