DNA polymerase-primase from embryos of Drosophila melanogaster. The DNA polymerase subunit

The DNA polymerase-primase from Drosophila melanogaster has been separated into its constituent polymerase and primase subunits by sedimentation in glycerol gradients containing 50% ethylene glycol. Both activities have been obtained in good yield. The properties of the 182-kDa polymerase subunit are similar to those of the intact four-subunit enzyme. However, there are three significant differences. (i) The polymerase activity of the 182-kDa subunit shows an increased thermolability; (ii) the pause sites during replication of singly primed, single-stranded circular DNA by the 182-kDa subunit are altered; and (iii) unlike the intact enzyme, the 182-kDa subunit is highly processive in the presence of the single-stranded DNA-binding protein of Escherichia coli.     

DNA polymerase-primase from embryos of Drosophila melanogaster. DNA primase subunits

The primase associated with the DNA polymerase-primase of Drosophila melanogaster fails to show enzymatic turnover. However, it does show turnover when dissociated from the intact polymerase-primase. Both forms of the enzyme can catalyze the synthesis of primers that are not complementary to the DNA template. Like the intact enzyme, the isolated primase synthesizes primers of a unique chain length; however, they are twice as long as those synthesized by the polymerase-primase. The activity of the primase separated from the polymerase-primase is similar in all other respects to the intact polymerase-primase.     

Interaction of ribonuclease H from Drosophila melanogaster embryos with DNA polymerase-primase

An RNase H was purified 2,500-fold to near homogeneity from early embryos of Drosophila melanogaster. The purified enzyme has an approximate molecular weight of 180,000 and appears to consist of two 49,000- and two 39,000-dalton polypeptides. The enzyme specifically hydrolyzes RNA.DNA hybrids and releases oligoribonucleotides ranging in size from 2-9 residues. The RNase H can also remove RNA primers that are synthesized and subsequently elongated by the Drosophila polymerase-primase. Preincubation of the RNase H from D. melanogaster embryos with the homologous DNA polymerase-primase results in an increased rate of DNA synthesis. The DNA chains synthesized under these conditions are shorter than those synthesized in the absence of the RNase H, and the rate of primer synthesis is increased significantly. These findings suggest that the RNase H forms a complex with the polymerase-primase, increasing its recycling capacity and thereby increasing the frequency of chain initiation.     

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

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

On the fidelity of DNA replication. Isolation of high fidelity DNA polymerase-primase complexes by immunoaffinity chromatography

Error rates for conventionally purified DNA polymerase-alpha from calf thymus, chicken, and human sources have been reported to be one in 10,000 to one in 40,000 nucleotides incorporated. Isolation of polymerase-alpha by immunoaffinity chromatography yields a multiprotein high molecular weight replication complex that contains an associated DNA primase (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We have isolated DNA polymerase-primase complexes from calf thymus, from a human lymphoblast cell line (TK-6), and from Chinese hamster lung cells (V-79) using two different methods of immunoaffinity chromatography. These enzyme complexes are 12- to 20-fold more accurate than conventionally purified calf thymus DNA polymerase-alpha when assayed using the phi X174am3 fidelity assay; estimated error rates are one in 460,000 to one in 830,000 nucleotides incorporated when the enzyme complex is freshly isolated. The polymerase-primase complex from calf thymus exhibited no detectable 3'----5' exonuclease activity using a heteroduplex substrate containing a single 3'-terminal mismatched nucleotide. Upon prolonged storage at -70 degrees C, the error rate of the immunoaffinity-purified calf thymus DNA polymerase-primase complex increases to about one in 50,000 nucleotides incorporated, an error rate similar to that exhibited by conventional isolates of DNA polymerase-alpha.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.     

The DNA synthesizing subunit of polymerase-primase from calf thymus.

The DNA synthesizing subunit (alpha-subunit) of DNA polymerase-alpha from calf thymus was separated from the other three subunits by immunoaffinity chromatography. The enzymatic properties of the alpha-subunit were characterized and compared with those of the four-subunit complex. Free alpha-subunit behaved in many respects like the four-subunit polymerase-primase. It was inhibited by aphidicolin and butylanilino-deoxyATP and catalyzed DNA synthesis on both gapped duplex DNA as well as primed single-stranded DNA with a preference of gapped DNA. The alpha-subunit is a quasi-processive enzyme with a processivity for about 9 nucleotides incorporated per single primer binding event. This is 2-fold lower than the processivity of the four-subunit complex. Despite this moderate processivity, free alpha-subunit was able to synthesize long stretches of DNA on singly primed natural psi X174am16 DNA. The accuracy of DNA synthesis of the free alpha-subunit was determined by using the psi X174am16 reversion assay to be 1 error per 50,000 nucleotides incorporated. An in vitro accuracy of 1 error in 54,000 nucleotides incorporated was measured in parallel for the four-subunit complex. Thus, the smaller subunits do not contribute to the overall accuracy of DNA polymerase-alpha. Consistent with this result is the observation that the polymerase to 3'----5'-exonuclease ratio was less than 1 to 2,500,000. Therefore, there is no evidence for the action of a cryptic proofreading activity with the alpha-subunit of DNA polymerase-alpha of mammalian origin.     

Delta-type DNA polymerase characterized from Drosophila melanogaster embryos.

Genetic and biochemical evidence suggests there are at least three DNA polymerases required for replication in eukaryotic cells. However, Drosophila embryonic cells have a very short duration S phase which is regulated differently. To address the question of whether embryos utilize different DNA polymerases, we employed Mono Q anion exchange chromatography to resolve the DNA polymerase activities. Two types of DNA polymerase, DNA polymerase delta and DNA polymerase alpha, were distinguished by: 1. copurification of DNA primase or 3'-5'exonuclease activities; 2. immunoblot analysis with alpha-specific polyclonal antisera; 3. sensitivity to aphidicolin and BuPdGTP; and 4. processivity measurements with and without Proliferating Cell Nuclear Antigen. These observations suggest that Drosophila embryos, similar to nonembryonic cells, have both alpha- and delta-type DNA polymerases.     

T4 DNA polymerase: Rates and processivity on single-stranded DNA templates

Three different methods have been used to determine the rate at which an individual bacteriophage T4 DNA polymerase molecule moves when synthesizing DNA on a single-stranded DNA template chain. These methods agree in suggesting an in vitro rate for this enzyme of about 250 nucleotides per second at 37 °C. This rate is close to the rate at which bacteriophage T4 replication forks move in vivo (about 500 nucleotides per second). Comparison with the overall amount of DNA synthesis seen in in vitro reactions reveals that only a small fraction of the T4 DNA polymerase molecules present are synthesizing DNA at any one time. This is explicable in terms of the limited processivity of the enzyme in these reactions, along with its capacity for non-productive DNA binding to the DNA template molecules.     

Apurinic DNA endonucleases from Drosophila melanogaster embryos

Summary Apurinic DNA endonuclease activity from Drosophila melanogaster embryos was resolved into two separable forms by phosphocellulose chromatography, one which flowed through the column (Fraction I) and the other which was retained and eluted at approximately 200 mM potassium phosphate (Fraction II). Both fractions, purified further by glycerol gradient sedimentation, were found to introduce nicks into DNA that were specific for and equal in number to the alkali-labile sites in depurinated DNA. They had similar apparent K_m values for apurinic sites (0.7 nM apurinic sites for Fraction I and 0.8 nM for Fraction II), but differed with respect to optimal pH, Mg⁺⁺ requirement and sensitivity to EDTA.     

DNA polymerase alpha from Drosophila melanogaster embryos. Subunit structure

The homogeneous DNA polymerase alpha from early embryos of Drosophila melanogaster contains four polypeptides designated alpha, beta, gamma, and delta, with molecular weights of 148,000, 58,000, 46,000, and 43,000, respectively (Banks, G. R., Boezi, J. A., and Lehman, I. R. (1979) J. Biol. Chem. 254, 9886-9892). The four polypeptides are structurally distinct from one another, as indicated by their different peptide patterns following limited proteolysis with Staphylococcus aureus protease. Furthermore, the inclusion of the protease inhibitors, leupeptin and pepstatin, in addition to phenpylmethylsulfonyl fluoride and sodium metabisulfite, which are used routinely during the purification, does not alter the pattern of polypeptides in the purified polymerase, suggesting that the four polypeptides are not a consequence of nonspecific proteolysis during purification. Thus, the alpha, beta, gamma, and delta polypeptides appear to be distinct subunits of the alpha-DNA polymerase of D. melanogaster. The alpha subunit is required for DNA polymerase activity. However, the specific activity of the isolated subunit is substantially lower than when it is associated with the beta, gamma, and delta subunits.     

DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization

A DNA ligase has been purified approximately 2,100-fold, to near-homogeneity, from Drosophila melanogaster 6-12-h embryos and was shown to catalyze the formation of 3',5'-phosphodiester bonds. Polypeptides with molecular weights 83,000, 75,000, and 64,000 were observed when the purified enzyme was electrophoresed under denaturing conditions. These polypeptides were shown by partial proteolysis studies and two-dimensional gel analysis to be structurally related. The two smaller polypeptides were presumably derived from the largest, 83,000 molecular weight protein, by proteolysis during purification or in vivo. All three polypeptides formed enzyme-adenylylate complexes in the absence of DNA. Drosophila DNA ligase had a Stokes radius of 45 A, a sedimentation coefficient of 4.3 S, and a frictional ratio of 1.6, yielding a calculated molecular weight of 79,800. These studies indicate that DNA ligase from Drosophila embryos is a monomer. The purified ligase was free of detectable ATPase, nuclease, topoisomerase, and DNA polymerase activities. The enzyme exhibited an absolute requirement for ATP in the joining reaction. A divalent metal was required and N-ethylmaleimide inhibited the reaction. Formation of phosphodiester bonds by Drosophila ligase required the presence of 5'-phosphoryl and 3'-hydroxyl termini. The purified enzyme restored biological activity to endonucleolytically cleaved pBR322 DNA. The specific activity of Drosophila DNA ligase was highest in unfertilized eggs. Developing embryos had 5-10-fold more ligase activity than at any later time in development.     

In vitro production of large single-stranded templates for DNA sequencing.

A method has been developed for the preparation of large single-stranded DNA sequencing templates from primary cloning plasmids or cosmids. The method utilizes the separate action of T7 Gene 6 exonuclease and exonuclease III to generate large quantities of single-stranded template for each strand of a large-cloned fragment. Since the procedure is intended for use on primary clones, it avoids the time-consuming subcloning steps associated with most sequencing programs. The procedure also has the advantage of avoiding clone instability problems associated with subcloning in M13.     

Initiation of bacteriophage PRD1 DNA replication on single-stranded templates

In vitro studies have demonstrated that single-stranded DNA molecules containing the 3' terminal nucleotides of the PRD1 DNA replication origin can support initiation by a protein-primed mechanism. We have determined the minimal requirements for priming by analyzing the template activity of various deletion derivatives. Our results showed that the 3'-terminal 15 nucleotides of the replication origin are sufficient for priming. The finding that the requirements for recognition of replication origin are different from those for priming suggests that there are two distinct steps during initiation of PRD1 DNA replication: first, recognition of the replication origin on double-stranded DNA and second, the priming event on single-stranded DNA. In addition our results showed that additional bases at the 3' end of templates did not affect priming activity, suggesting that the priming site is searched for from inside the template.     

Isolation of a single-stranded extrachromosomal DNA from germinating wheat embryos

Newly-synthesized cytoplasmic non-mitochondrial DNA was isolated from wheat embryos which had been germinated in the presence of [¹⁴C]-thymidine for a time period not long enough to trigger the first post-dormant round of the nuclear DNA replication. This extrachromosomal DNA fraction consisted of linear single-stranded polydeoxyribonucleotide chains, corresponding in size to approximately 1.1×10⁶ daltons, and amounted to about 0.5% of the total cellular DNA content. It is suggested that the appearance of the newly-synthesized polydeoxyribonucleotide chains in the cytoplasm may be a physiological signal for the initiation of the nuclear DNA replication in germinating wheat embryo cells.     

Two distinct DNA ligases from Drosophila melanogaster embryos

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The Km for ATP is 2.7 X 10(-6) M for DNA ligase I, and 3.0 X 10(-5) M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0-30 mM KCl), but DNA ligase II is active at high salt concentrations (50-100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637-10645] seem to be the same enzyme.