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.

Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy.

The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.

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.

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.

Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-thymine glycol.

Human DNA polymerase N (POLN or pol nu) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiation-induced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.

Transcription factor YB-1 mediates DNA polymerase alpha gene expression.

Y-box protein-1 involvement in cyclin A and B1 gene regulation has recently been demonstrated. A more generalized role of this protein for cell replication is hypothesized as numerous regulatory sequences of cell cycle-related genes contain putative binding sites. In the present study the DNA polymerase alpha (DPA) gene is identified as another YB-1-responsive gene with a Y-box and 3' inverted repeat sequence, designated DPA RE-1, in the serum-responsive promoter region. Overexpressed YB-1 concentration-dependently trans-activated DPA gene expression in reporter assays and Southwestern blotting as well as DNA binding analyses revealed binding of distinct endogenous proteins to the RE-1 with molecular sizes of 26, 32 and 52 kDa. Among these, YB-1 binding was confirmed using recombinant as well as endogenous proteins, with preferential single-stranded DNA binding. Early serum growth response in mesangial cells was accompanied by a nuclear YB-1 shift and nucleocomplex formation at the RE-1. Fine mapping of the DPA RE-1 sequence unraveled a dependence on co-factors for trans-regulation with gene activation in the context of a heterologous SV40 promoter but suppression in the context of the abbreviated homologous promoter sequence. A YB-1 knock down resulted in decreased DPA transcription rates and abrogated the serum-dependent induction of DPA transcription. These results link YB-1 with serum responsiveness of DPA gene expression and provide insight into the required sequence and protein binding context.

[Effects of cyclosporine A on NIT-1 beta cell proliferation and pol alpha1 gene expression in vitro].

OBJECTIVE: To investigate the effects of cyclosporine A on the proliferation and pol alpha1 mRNA expression of cultured NIT-1 beta cells. METHODS: After exposure to cyclosporine A at various concentrations (0.05 to 10 micromol/L) for 48 h and 72 h, NIT-1 cell proliferation was analyzed by MTT assay and the gene expression determined by reverse transcriptional PCR (RT-PCR). RESULTS: Forty-eight-hour and 72-hour cyclosporine A exposure inhibited the cell proliferation in a concentration- dependent manner, and at the concentration of 10 micromol/L, cyclosporine A also decreased pol alpha1 mRNA expression after a 48-hour exposure. CONCLUSION: Cyclosporine A can effectively inhibit the proliferation of NIT-1 cells possibly through down-regulating the expression of pol alpha1 mRNA.

Interaction of DNA polymerase I (Klenow fragment) with the single-stranded template beyond the site of synthesis.

Cocrystal structures of DNA polymerases from the Pol I (or A) family have provided only limited information about the location of the single-stranded template beyond the site of nucleotide incorporation, revealing contacts with the templating position and its immediate 5' neighbor. No structural information exists for template residues more remote from the polymerase active site. Using a competition binding assay, we have established that Klenow fragment contacts at least the first four unpaired template nucleotides, though the quantitative contribution of any single contact is relatively small. Photochemical cross-linking indicated that the first unpaired template base beyond the primer terminus is close to Y766, as expected, and the two following template bases are close to F771 on the surface of the fingers subdomain. We have constructed point mutations in the region of the fingers subdomain implicated by these experiments. Cocrystal structures of family A DNA polymerases predict contacts between the template strand and S769, F771, and R841, and our DNA binding assays provide evidence for the functional importance of these contacts. Overall, the data are most consistent with the template strand following a path over the fingers subdomain, close to the side chain of R836 and a neighboring cluster of positively charged residues.

Replicative enzymes and ageing: importance of DNA polymerase alpha function to the events of cellular ageing.

A hallmark of cellular ageing is the failure of senescing cells to initiate DNA synthesis and transition from G1 into S phase of the cell cycle. This transition is normally dependent on or concomitant with expression of a set of genes specifying cellular proteins, some of which directly participate in DNA replication. Deregulation of this gene expression may play a pivotal role in the ageing process. The number of known enzymes and co-factors required to maintain integrity of the genome during eukaryotic DNA replication has increased significantly in the past few years, and includes proteins essential for DNA replication and repair, as well as for cell cycle regulation. In eukaryotic cells, ranging from yeast to man, a replicative enzyme essential for initiation of transcription is DNA polymerase alpha (pol alpha), the activity of which is coordinately regulated with the initiation of DNA synthesis. DNA pol alpha, by means of its primase subunit, has the unique ability to initiate de novo DNA synthesis, and as a consequence, is required for the initiation of continuous (leading-strand) DNA synthesis at an origin of replication, as well as for initiation of discontinuous (lagging-strand) DNA synthesis. The dual role of the pol alpha-primase complex makes it a potential interactant with the regulatory mechanisms controlling entry into S phase. The purpose of this review is to address the regulation and/or modulation of DNA pol alpha during ageing that may play a key role in the cascade of events which ultimately leads to the failure of old cells to enter or complete S phase of the cell cycle.

Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2.

The transcription factor E2F-1 plays a pivotal role in the regulation of G1/S transition in higher eukaryotes cell cycle. We used a cell line containing an inducible E2F-1 and oligonucleotide microarray analysis to identify novel E2F target genes. We show that E2F-1 up-regulates the expression of a number of genes coding for components of the DNA replication machinery. Among them is the gene coding for the 32 Kd subunit of replication protein A (RPA2). Replication protein A is the most abundant single strand DNA binding complex and it is essential for DNA replication. We demonstrate that RPA2 is a novel E2F target gene whose expression can be directly regulated by E2F-1 via E2F binding sites in its promoter. In addition, expression of Topoisomerase IIalpha and subunit IV of DNA polymerase alpha is also up-regulated upon E2F-1 induction. Taken together, these results provide novel links between components of the DNA replication machinery and the cell growth regulatory pathway involving the Rb tumor suppressor and E2F.

Molecular cloning of a gene (poIA) coding for an unusual DNA polymerase I from Treponema pallidum.

The gene coding for the DNA polymerase I from Treponema pallidum, Nichols strain, was cloned and sequenced. Depending on which of the two alternative initiation codons was used, the protein was either 997 or 1015 amino acids long and the predicted protein had a molecular mass of either 112 or 114 kDa. Sequence comparisons with other polA genes showed that all three domains expected in the DNA polymerase I class of enzymes were present in the protein (5'-3' exonuclease, 3'-5' exonuclease and polymerase domains). Additionally, there were four unique insertions of 20-30 amino acids each, not seen in other DNA polymerase I enzymes. Two of the inserts were near the boundary of the two exonuclease domains and the other two interrupted the 3'-5' exonuclease domain which is involved in proofreading. The predicted amino-acid sequence had an exceptionally high content of cysteine (2.4% compared with <0.05% for most other sequenced DNA polymerase I enzymes). The polA gene was further cloned into pProEXHTa for expression and purification. The transformants expressed a protein of 115 kDa. Antibodies raised against synthetic peptide fragments of the putative DNA polymerase I recognised the 115-kda band in Western blot analysis. No DNA synthesis activity could be demonstrated on a primed single-stranded template. Although significant quantities of the protein were produced in the host Escherichia coli carrying the plasmid, it was not capable of complementing a polA(-) mutant in the replication of a polA-dependent plasmid.

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.

Ara-C affects formation of cancer cell DNA synthesome replication intermediates.

PURPOSE: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. METHODS: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta). RESULTS: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug. CONCLUSIONS: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.

Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes.

Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair. The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes. This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs. Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Elevated expression of DNA polymerase beta gene in glioma cell lines with acquired resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea.

We investigated the expression of DNA polymerase beta (beta-pol) and O6-methylguanine-DNA methyltransferase (MGMT) in human glioma cells with acquired resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoure a (ACNU) and in the parent cells. ACNU-resistant T430 (T430R) and A172 (A172R) glioma cell lines were established following repeated exposure to ACNU. The level of MGMT mRNA expression was elevated in T430R, but not in A172R. In contrast, the level of beta-pol mRNA expression and the level of beta-pol protein were elevated in A172R, compared with the parent cells. While the mechanism of MGMT repair has been considered to be important in the drug resistance of human brain tumors to ACNU, our present results demonstrate that beta-pol may also play an important role in the acquisition of tumor cell resistance to ACNU in human gliomas.

Potential role of DNA polymerase beta in gene therapy against cancer: a case for colorectal cancer.

Genetic instability characterized by the accumulation of mutations of tumor suppressor genes and oncogenes appears to be associated with carcinogenesis in colorectal and other cancers. Mutations of DNA polymerase beta (pol beta) and related chromosomal alterations appear to be consistent with the causal role of a "mutator phenotype' in carcinogenesis. However, homozygous knockout pol beta mutations appear to interfere with embryogenesis. Increased pol beta activity (i.e. relative to pol alpha activity) has been associated with cell cycle arrest. The related aphidicolin-resistant DNA replication has been observed primarily in differentiating cells, including the mammalian blastocyst, adrenal cortex, thyroid, anterior pituitary, and the mechanism of endoreduplication (amitotic over-replication of DNA) can be traced to lower eukaryotes. This increased activity in relation to terminal commitment is inconsistent with a simple "DNA repair' view of pol beta. It is therefore proposed that pol beta may play a more fundamental role in cellular differentiation through involvement in a putative subgenomic DNA replication-based model of terminal gene expression. Thus genetic instability, loss of differentiation, and carcinogenesis may result from aberration(s) or "derailment' of such replication-based mechanism of terminal gene expression. It is suggested to examine the relationship of DNA pol beta to genomic instability and carcinogenesis using genetic analyses and antisense technology with possible applications for gene therapy against colorectal cancer.

Effect of different vitamin A status on carcinogen-induced DNA damage and repair enzymes in rats.

The effect of different vitamin A status on events following DNA damage by hepatocarcinogens was investigated in rats. Formation of single-strand breaks in nuclear DNA induced by aflatoxin B1 and N-nitrosodimethylamine was observed to be more pronounced after vitamin A-deficiency. This enhanced damage was reversed upon vitamin A supplementation. Subsequent to DNA damage, the induction of repair enzymes poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was found to be significantly higher in vitamin A-deficient rats. Vitamin A supplementation brought down the induction to the levels found in rats maintained on normal diet. Vitamin A thus may control carcinogenesis by manipulating molecular events at the initiation stage.

Construction and expression of a chimeric gene encoding human terminal deoxynucleotidyltransferase and DNA polymerase beta.

A domain substitution experiment was carried out between the structurally related DNA-polymerizing enzymes Pol beta and TdT to investigate the region of Pol beta required for template utilization. Site-directed mutagenesis and recombinant DNA procedures were used for construction of a gene encoding a chimeric form of the two enzymes and termed TDT::POLB, in which the DNA region encoding amino acids (aa) 154-212 of TdT was replaced by the corresponding region encoding aa 1-60 of POL beta. The construction was confirmed by restriction analysis and DNA sequencing. Since this region of POL beta represents most of the N-terminal domain of the enzyme possessing single-stranded DNA (ssDNA)-binding activity, it was hypothesized that the chimeric protein, unlike TdT, might possess template-dependent DNA polymerase activity. The chimeric gene product was produced in Escherichia coli, purified and subjected to preliminary enzymological characterization. The finding that the chimeric TdT::Pol beta protein possessed significant template-dependent polymerase activity suggests that aa 1-60 of Pol beta are involved in template utilization during the polymerization reaction, as suggested by the previous finding that the 8-kDa N-terminal domain of Pol beta possesses ssDNA-binding activity [Kumar et al., J. Biol. Chem. 265 (1990a) 2124-2131; Kumar et al., Biochemistry 29 (1990b) 7156-7159; Prasad et al., J. Biol. Chem. 268 (1993) 22746-22755].

The bovine DNA polymerase beta promoter: cloning, characterization and comparison with the human core promoter.

The core promoter of the human DNA polymerase beta (beta Pol)-encoding gene (POL beta) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755-12763]. To study the homologous promoter (ppol beta) in a bovine system, we cloned and characterized the 5'-flanking region of the bovine gene (pol beta). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5'-flanking region was isolated from a testis library in bacteriophage lambda EMBL3. S1 nuclease mapping and primer extension analysis of the 5'-end of the pol beta mRNA identified the major transcription start point (tsp), which is located 142-bp 5' of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5'-deletion mutants demonstrated that a fragment of only 91-bp 5' of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOL beta). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Sp1-binding sites, and the spacing separating them.

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.