Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.     

An endogenous protein inhibitor of DNA polymerase alpha in normal and neoplastic rat mammary tissues

Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.     

DNA primase activity found in an alpha-like DNA polymerase obtained from Halobacterium halobium

An aphidicolin-sensitive DNA polymerase was purified from extracts of Halobacterium halobium. The analysis of this alpha-like DNA polymerase on polyacrylamide gels under denaturing conditions revealed two peptides with molecular masses of 70 kDa and 60 kDa in equal amounts. Like the DNA polymerase alpha isolated from eukaryotes, the alpha-like DNA polymerase possesses primase activity using UTP and polydeoxyadenylate as template. The primase activity was sensitive to aphidicolin and inhibited by an antiserum against the alpha-like DNA polymerase of H. halobium. The primase activity was dependent on the presence of high salt concentrations.     

DNA polymerases from Chlamydomonas reinhardii. Purification and properties.

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. 'Activated' calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.     

A DNA polymerase alpha partially resistant to aphidicolin in cells and embryos of Med-fly (Ceratitis capitata Wied.)

We have studied the DNA polymerase activities in cultured cells and embryos of Med-fly (Ceratitis capitata Wied.) and we observed that only DNA polymerases alpha and gamma were detectable in crude extracts. The level of DNA polymerase alpha, measured during the life cycle of the insect embryos, increased in parallel with the rate of embryonic cell proliferation, whereas DNA polymerase gamma increased at much later fertilization time, when cell differentiation is already taking place. DNA polymerase alpha, purified 100 folds from Med-fly embryos, was 10 times more resistant to aphidicolin, its specific inhibitor, than the mammalian DNA polymerase alpha. In situ visualization of the active peptides after NaDodSO4/PAGE, confirmed that only high Mr DNA polymerase fragments were present in embryo extracts and in purified preparations of DNA polymerase alpha. It appears that C. capitata cells represent a rather peculiar system in the phylogeny of DNA polymerases since they are devoid of DNA polymerase beta and present a DNA polymerase alpha partially resistant to aphidicolin.     

A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure.

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 X 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 X 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.     

Transfer of the Epstein-Barr virus genes coding for small RNAs to human lymphoid cells with a vector carrying a dominant selectable marker.

Epstein-Barr virus (EBV)-negative, Burkitt-like lymphoma-derived cells were transformed with a transducing vector (pSV2-gpt) containing the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase (XGPRT) and with a derivative of PSV2-gpt that carries the genes for the EBV-associated small RNAs on the EcoRI J fragment of B95-8 EBV DNA inserted at the unique EcoRI site (pJ-gpt). Cells transformed with PSV2-gpt and pJ-gpt express the E. coli gpt gene to approximately the same extent, judged by determinations of the XGPRT activity of cell extracts. Blot hybridisation experiments with restriction endonuclease-cleaved DNA from the transformants have revealed the presence of vector DNA sequences in the cells, at least some of which are most probably integrated into high mol. wt. chromosomal DNA. Northern blot hybridisation analysis of cytoplasmic RNA from pJ-gpt-transformed cells revealed the presence of an EcoRI J DNA complementary RNA species of the same size as the EBV DNA-encoded small RNAs found in EBV-transformed cells.     

The 'light' and 'medium' subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis: isolation of the genes, nucleotide and amino acid sequence

The 'light' (L) and the 'medium' (M) subunits of the photosynthetic reaction centre from Rhodopseudomonas viridis were isolated and their amino-terminal sequences, as well as the sequences of several chymotryptic peptides, determined. Rps. viridis DNA was cloned in the Escherichia coli plasmid pBR322. Mixed oligonucleotide probes derived from the amino acid sequences were synthesized and utilised to isolate one clone which contained the genes for the L and M subunits of the reaction centre as well as the alpha and beta subunits of the light-harvesting complex and part of the gene for the reaction centre cytochrome. The nucleotide sequences of the L and M subunit genes and teh derived amino acid sequences are presented. The L subunit consists of 273 amino acids and has a mol. wt of 30 571. The M subunit consists of 323 amino acids and has a mol. wt of 35 902. The primary structure is discussed in the light of the recently published secondary and tertiary structure which has shown that both subunits contain five membrane-spanning helices.     

Expression of active rat DNA polymerase beta in Escherichia coli

A recombinant plasmid for expression of rat DNA polymerase beta was constructed in a plasmid/phage chimeric vector, pUC118, by an oligonucleotide-directed mutagenesis technique. The insert contained a 1005 bp coding sequence for the whole rat DNA polymerase beta. The recombinant plasmid was designed to use the regulatory sequence of Escherichia coli lac operon and the initiation ATG codon for beta-galactosidase as those for DNA polymerase beta. The recombinant clone, JMp beta 5, obtained by transfection of E. coli JM109 with the plasmid, produced high levels of DNA polymerase activity and a 40-kDa polypeptide that were not detected in JM109 cell extract. Inducing this recombinant E. coli with isopropyl beta-thiogalactopyranoside (IPTG) yielded amounts of 40-kDa polypeptide as high as 19.3% of total protein. Another recombinant clone, JMp beta 2-1, which was constructed by an oligonucleotide-directed mutagenesis to use the second ATG codon for the initiation codon, thus deleting the first 17 amino acid residues from the amino terminus, produced neither high DNA polymerase activity nor the 40-kDa polypeptide. The evidence suggests that this amino-terminal structure is important for stability of this enzyme in E. coli. The DNA polymerase was purified to homogeneity from the IPTG-induced JMp beta 5 cells by fewer steps than the procedure for purification of DNA polymerase beta from animal cells. The properties of this enzyme in activity, chromatographic behavior, size, antigenicity, and also lack of associated nuclease activity were indistinguishable from those of DNA polymerase beta purified from rat cells, indicating the identity of the overproduced DNA polymerase in the JMp beta 5 and the rat DNA polymerase beta.     

A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader

The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.     

Characteristic alteration in the nuclear DNA polymerase activity during the cell division cycle of Saccharomyces cerevisiae

Specific activity of the intranuclear DNA polymerase in cdc-mutant cells of Saccharomyces cerevisiae was found to be characteristically changed by arrest in their specific stage of cell division cycle without a notable alteration in the total cellular activity. The activities were low in the nuclei of cdc 25, cdc 28 and cdc 4, which were arrested in early to mid G1 phase by temperature shift-up, and in the nuclei of wild-type cells (A364A), which were arrested in early G1 phase by alpha-factor treatment, while high level of the activity was found in the nuclei of cdc 7 and cdc 8, which were arrested at late G1 and S phase, respectively. Activity-gel analysis of DNA polymerase in the nuclear extracts revealed the presence of two active peptides (120K and 72K), and the characteristic decrease in both active peptides was induced by arrest in early to mid G1 phase. Consequently, it is strongly suggested that intranuclear DNA polymerase activity alters in a dependent fashion on progression of cell division cycle. Subunit analysis indicated that the purified DNA polymerase I is constructed from two subunit peptides of 120K and 62K, and the large subunit possesses catalytic activity.     

Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition.

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.     

Structure of the N-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase

We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation. This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS. The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT). However, the orientation of the two domains in AT-N440 differs from that in KNT. The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase. AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases. The location of residues conserved in all ATase sequences was found to cluster around the active site. Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.     

Cytochrome P-450 in culture forms of Trypanosoma cruzi

Trypanosoma cruzi epimastigote and trypomastigote forms contain microsomal peptides in the 40-60,000 mol. wt region, some of which are heme-staining-positive and are induced by phenobarbital, as indicated by SDS-gel electrophoresis and by double-labeling experiments. Epimastigotes show induced peptides of mol. wt 56,000, 52,000, 49,000, 44,000, 42,000 and 40,500 whereas only one peptide (52,500 mol. wt) is increased in trypomastigotes. Fractionation of microsomes derived from epimastigotes by octylamine Sepharose-4B column chromatography reveals the presence of two heme peptides with mol. wt of 55,800 and 56,600. The pooled fraction has a typical cytochrome P-450 CO-difference spectrum and appears to correspond to a high spin form. The demonstration of the existence of this family of hemoproteins in T. cruzi further supports the idea that resistance to chemotherapeutic agents is due to active metabolism. The active metabolism, however, may not be similar in the various developmental forms of this organism since differences exist in the patterns of induction of heme-positive microsomal peptides.     

Sequential changes in DNA polymerases alpha and beta during diethylnitrosamine-induced carcinogenesis

It has often been suggested that the high molecular weight DNA polymerase alpha of eukaryotes plays a role in de novo replication of DNA, while the low molecular weight polymerase beta is involved in repair replication. Previous studies have shown that when diethylnitrosamine is fed in the diet to rats it causes after a few weeks an increase in de novo replication of DNA, which then returns to normal values. In contrast, repair replication may be expected to continue throughout the feeding period. Study of DNA polymerase activity in livers of animals during carcinogenesis showed that an increase in polymerase alpha occurred at the time of increased de novo replication, while there was a gradual increase in polymerase beta during the time diethylnitrosamine was present in the diet. When diethylnitrosamine treatment was stopped, there was a rapid drop in polymerase beta activity. These results support the view that the polymerase alpha is involved in DNA replication, that the polymerase beta functions in repair replication, and that the beta enzyme can be induced by chronic damage to DNA.     

The mammalian primase is part of a high molecular weight DNA polymerase alpha polypeptide

The recently discovered eukaryotic primases have been found in tight association with certain DNA polymerase alpha forms. Here I present evidence that the high mol. wt. catalytic polypeptide (125,000) of an apparently homogeneous DNA polymerase alpha from freshly harvested calf thymus contains both polymerase and primase activity. This conclusion derives from the following three facts: (1) the two enzyme activities cannot be separated upon velocity sedimentation in 1.7 M urea, (2) both activities elute at a pI of 5.25 upon chromatofocussing and (3) after SDS-electrophoresis, renaturation of the enzymes in situ and measurement of DNA polymerase and primase activities in the gels, both enzymes have identical mobilities and coincide with the high mol. wt. catalytic subunit of DNA polymerase alpha.