DNA synthesis in isolated mitochondria and mitochondrial extracts from wheat embryos

DNA synthesis was studied using purified wheat embryo mitochondria as well as mitochondrial lysates deprived of endogenous DNA. The optimal conditions for DNA synthesis are very similar in both systems: ATP stimulates dramatically mitochondrial DNA synthesis and magnesium is a better co-factor than manganese, contrary to what has been reported in animal mitochondrial systems. Wheat mitochondrial DNA synthesis is resistant to aphidicolin and strongly inhibited by dideoxythymidine triphosphate and ethidium bromide. Thus, the DNA polymerase involved in this system seems to be the same as that previously purified and characterized from wheat embryo mitochondria (Christopheet al., Plant Science Letters 21: 181, 1981). Two different approaches: restriction endonuclease digestion followed by electrophoresis, and autoradiography and cesium chloride equilibrium centrifugation of mitochondrial DNA, where BrdUTP has been incorporated instead of TTP, show that long stretches of the mitochondrial genome have been synthesized.     

Adenosine 5'-O-(3-thiotriphosphate) can support the formation of an initiation complex between the DNA polymerase III holoenzyme and primed DNA

Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) will substitute for ATP in the formation of an initiation complex between the DNA polymerase III holoenzyme of Escherichia coli and primed DNA. The initiation complex formed in the presence of ATP gamma S between the DNA polymerase III holoenzyme and single-stranded DNA-binding protein-encoated primed M13 Gori DNA is stabile and isolable by gel filtration at room temperature. Upon addition of the four required deoxynucleoside triphosphates, this complex is rapidly converted to the duplex replicative form without dissociation of the polymerase. Initiation complexes formed in the presence of either ATP gamma S or ATP are indistinguishable by their resistance to antibody directed against the beta subunit of the holoenzyme and by their ability to elongate without further activation. A 2-fold difference was observed, however, in both the extent of initiation complex formation and in the dissociation of initiation complexes once formed. This difference is discussed in the light of previous proposals regarding a dimeric polymerase capable of replicating both strands at a replication fork concurrently.     

DNA primase associated with 10 S DNA polymerase alpha from calf thymus

Among multiple subspecies of DNA polymerase alpha of calf thymus, only 10 S DNA polymerase alpha had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase alpha through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase alpha. These results indicate that the primase is tightly bound to 10 S DNA polymerase alpha. The RNA polymerizing activity was resistant to alpha-amanitin, required high concentration of all four ribonucleoside triphosphates (800 microM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase alpha because it was strongly inhibited by araCTP, resistant to d2TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

Phosphatidylinositol-dependent activation of DNA polymerase alpha

DNA polymerase alpha was activated in vitro by cAMP-independent, phospholipid-dependent, protein kinase catalytic subunit. Of the phospholipids examined, phosphatidylinositol showed the greatest potential for interaction with protein kinase and ATP to activate DNA polymerase alpha in vitro. DNA polymerase alpha was directly activated by phosphorylated phosphatidylinositol in the absence of protein kinase and ATP. Activation of DNA polymerase alpha as a function of phosphorylation was demonstrated using 32P-ATP as the phosphate donor. In vitro treatment of the enzyme with phosphatidylinositol produced Linweaver-Burk plots showing noncompetitive kinetics of enzyme activation, suggesting that activation occurs prior to binding of the enzyme to DNA template/primer. These data indicate that DNA polymerase alpha may be activated in vitro in the presence of protein kinase, ATP, and phosphatidylinositol, and suggest that phosphorylation of the enzyme may constitute an intracellular mechanism of enzyme activation.     

An in vitro DNA phosphorothioate modification reaction

Phosphorothioation (PT) involves the replacement of a nonbridging phosphate oxygen on the DNA backbone with sulfur. In bacteria, the procedure is both sequence- and stereo-specific. We reconstituted the PT reaction using purified DndCDE from Salmonella enterica and IscS from Escherichia coli. We determined that the in vitro process of PT was oxygen sensitive. Only one strand on a double-stranded (ds) DNA substrate was modified in the reaction. The modification was dominant between G and A in the GAAC/GTTC conserved sequence. The modification between G and T required the presence of PT between G and A on the opposite strand. Cysteine, S-adenosyl methionine (SAM) and the formation of an iron-sulfur cluster in DndCDE (DndCDE-FeS) were essential for the process. Results from SAM cleavage reactions support the supposition that PT is a radical SAM reaction. Adenosine triphosphate (ATP) promoted the reaction but was not essential. The data and conclusions presented suggest that the PT reaction in bacteria involves three steps. The first step is the binding of DndCDE-FeS to DNA and searching for the modification sequence, possibly with the help of ATP. Cysteine locks DndCDE-FeS to the modification site with an appropriate protein conformation. SAM triggers the radical SAM reaction to complete the oxygen-sulfur swapping.     

DNA polymerase beta from brain neurons is a repair enzyme.

DNA polymerase beta was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian beta-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no alpha-polymerase, 99.2% beta-polymerase and only 0.8% gamma-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since beta-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the beta enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2',3'-dideoxyribosylthymine 5'-triphosphate (d2TTP), a potent beta-polymerase inhibitor. However, the presence of minute amounts of gamma-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase gamma in DNA repair. In view of the similarity of the neuronal DNA polymerase beta with all other mammalian beta-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all beta-polymerases.     

Effect of 2'',3''-dideoxythymidine-5''-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase alpha is the only polymerase required for cellular DNA replication.

We have studied the effects of the nucleotide analogue, 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) on replicative DNA synthesis in HeLa cell lysates. As previously demonstrated (1), such lysates carry out extensive DNA synthesis in vitro, at rates and in a fashion similar to in vivo DNA replication. We report here that all aspects of DNA synthesis in such lysates (total dNTP incorporation, elongation of continuous nascent strands, and the initiation, elongation, and joining of Okazaki pieces) are only slightly inhibited by concentrations of ddTTP as high as 100-500 micrometer when the dTTP concentration is maintained at 10 micrometer. This finding is consistent with the report by Edenberg, Anderson, and DePamphilis (2) that all aspects of replicative in vitro simian virus 40 DNA synthesis are also resistant to ddTTP. We also find, in agreement with Edenberg, Anderson, and DePamphilis (2), that DNA synthesis catalyzed by DNA polymerases beta or gamma is easily inhibited by ddTTP, while synthesis catalyzed by DNA polymerase alpha is very resistant. These observations suggest that DNA polymerase alpha may be the only DNA polymerase required for all aspects of cellular DNA synthesis.     

DNA primase associated with 10 S DNA polymerase α from calf thymus

Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d₂TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

S-adenosyl methionine alters the DNA contacts of the EcoKI methyltransferase.

The EcoKI methyltransferase methylates two adenines on opposite strands of its bipartite DNA recognition sequence AAC(N6)GTGC. The enzyme has a strong preference for hemimethylated DNA substrates, but the methylation state of the DNA does not influence its binding affinity. Methylation interference was used to compare the contacts made by the EcoKI methyltransferase with unmodified, hemimethylated or fully modified DNAs. Contacts were seen at or near the N7 position of guanine, in the major groove, for all of the guanines in the EcoKI recognition sequence, and at two guanines on the edge of the intervening spacer sequence. The presence of the cofactor and methyl donor S-adenosyl methionine had a striking effect on the interference pattern for unmodified DNA which could not be mimicked by the presence of the cofactor analogue S-adenosyl homocysteine. In contrast, S-adenosyl methionine had no effect on the interference patterns for either kind of hemimethylated DNA, or for fully modified DNA. Differences between the interference patterns for the unmodified DNA and any of the three forms of methylated DNA provide evidence that methylation of the target sequence influences the conformation of the protein-DNA interface, and illustrate the importance of S-adenosyl methionine in the distinction between unmodified and methylated DNA by the methyltransferase.     

Alterations in the phosphorylation and activity of DNA polymerase alpha correlate with the change in replicative DNA synthesis as quiescent cells re-enter the cell cycle

The regulation of DNA polymerase alpha was examined in quiescent, human fibroblast cells stimulated to re-enter the cell cycle by subculturing in fresh serum-containing medium. The level of DNA polymerase alpha activity was measured in cell lysates and after specific immunoprecipitation. DNA polymerase alpha activity increased approximately 10-fold during the period of measurement. The activity increase was coincident with an approximately 60-fold increase in thymidine incorporation in the whole cells representing the first S phase. The large increase in polymerase alpha activity was not predominantly the result of synthesis of new polymerase, since the abundance of the enzyme changed less than 2-fold over the measured period. The quantity of [32P]phosphate incorporated into two subunits (180 and 68 kilodaltons) of DNA polymerase alpha increased approximately 10-fold in parallel with the increase in polymerase activity. The specific activity of the cellular ATP pool remained nearly constant over the period of measurement, indicating that the increase in labeling reflects a true increase in incorporation of phosphate. Results from other laboratories indicate that phosphorylation of DNA polymerase alpha increases its catalytic activity. Our results then suggest that the activity increase observed in DNA polymerase alpha when quiescent, human fibroblasts are stimulated to proliferate is largely caused by a phosphorylation-dependent regulatory process.     

Replication of parvoviral DNA. I. Characterization of a nuclear lysate system.

We have developed a nuclear lysate system from infected, synchronized cells capable of synthesizing unit-length parvoviral DNA in vitro. It was necessary to supplement the nuclear lysates with the polyamines, spermidine and spermine, to prevent degradation of template and product DNAs. In this system RF, RI, and single-stranded progeny DNAs were synthesized. Label incorporated in viral RF DNA in vivo appeared first in RI DNA and then in single-stranded DNA during incubation in vitro. By sedimentation the product DNAs were identical to those found in infected cells. Their viral identity was confirmed by hybridization. The addition of ribonucleotides, RNase, or alpha-amanitin did not affect parvoviral DNA synthesis in this system. The results with the specific inhibitors of mammalian DNA polymerases, aphidicolin, N-ethylmaleimide, and 2',3'-dideoxythymidine 5'-triphosphate indicated that DNA polymerase alpha was required for synthesis of parvoviral DNA in the nuclear lysates. This requirement was confirmed by experiments with antibody to bovine DNA polymerase alpha.     

DNA polymerase I and DNA primase complex in yeast

Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I. Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract. Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity. Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction. Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex. Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity. These results suggest that these two activities exist as a complex and reside on the different polypeptides. Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs. The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs. Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity. The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin. The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose. Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2. Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.     

DNA replication in isolated HeLa cell nuclei

DNA replication was investigated in HeLa cell nuclei isolated from different phases of the cell cycle. DNA synthesis occurred only in S-phase nuclei and was dependent on the presence of the four deoxynucleoside triphosphates, Mg⁺⁺, ATP and S-phase cytoplasm. G₁-phase cytoplasm was unable to support such DNA synthesis. A purified preparation of calf thymus DNA polymerase, however, was able to replace S-phase cytoplasm in supporting ATP dependent DNA synthesis, which suggests that the S-phase cytoplasmic factor is a DNA polymerase. G₁-phase nuclei could under no conditions be stimulated to initiate DNA replication prematurely.     

Repair response of Escherichia coli to hydrogen peroxide DNA damage.

The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.     

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.     

DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction

We have used DNA footprinting techniques to analyze the interactions of five DNA replication proteins at a primer-template junction: the bacteriophage T4 DNA polymerase (the gene 43 protein), its three accessory proteins (the gene 44/62 and 45 proteins), and the gene 32 protein, which is the T4 helix-destabilizing (or single-stranded DNA-binding) protein. The 177-nucleotide-long DNA substrate consisted of a perfect 52-base pair hairpin helix with a protruding single-stranded 5' tail. As expected, the DNA polymerase binds near the 3' end of this molecule (at the primer-template junction) and protects the adjacent double-stranded region from cleavage. When the gene 32 protein binds to the single-stranded tail, it reduces the concentration of the DNA polymerase required to observe the polymerase footprint by 10-30-fold. Periodic ATP hydrolysis by the 44/62 protein is required to maintain the activity of the DNA polymerase holoenzyme (a complex of the 43, 44/62, and 45 proteins). Footprinting experiments demonstrate the formation of a weak complex between the DNA polymerase and the gene 45 protein, but there is no effect of the 44/62 protein or ATP on this enlarged footprint. We propose a model for holoenzyme function in which the complex of the three accessory proteins uses ATP hydrolysis to keep a moving polymerase tightly bound to the growing 3' end, providing a "clock" to measure polymerase stalling.     

Enhanced processivity of nuclear matrix bound DNA polymerase alpha from regenerating rat liver

Translocation of DNA during in vitro DNA synthesis on nuclear matrix bound replicational assemblies from regenerating rat liver was determined by measuring the processivity (average number of nucleotides added following one productive binding event of the polymerase to the DNA template) of nuclear matrix bound DNA polymerase alpha with poly(dT).oligo(A)10 as template primer. The matrix-bound polymerase had an average processivity (28.4 nucleotides) that was severalfold higher than the bulk nuclear DNA polymerase alpha activity extracted during nuclear matrix preparation (8.9 nucleotides). ATP at 1 mM markedly enhanced the activity and processivity of the matrix-bound polymerase but not the corresponding salt-soluble enzyme. The majority of the ATP-dependent activity and processivity enhancement was completed by 100 microM ATP and included products ranging up to full template length (1000-1200 nucleotides). Average processivity of the net ATP-stimulated polymerase activity exceeded 80 nucleotides with virtually all the DNA products greater than 50 nucleotides. Release of nuclear matrix bound DNA polymerase alpha by sonication resulted in a loss of ATP stimulation of activity and a corresponding decrease in processivity to a level similar to that of the salt-soluble polymerase (6.8 nucleotides). All nucleoside di- and triphosphates were as effective as ATP. Stimulation of both activity and processivity by the nonhydrolyzable ATP analogues adenosine 5'-O-(3-thiotriphosphate), 5'-adenylyl imidodiphosphate, and adenosine 5'-O-(1-thiotriphosphate) further suggested that the hydrolysis of ATP is not required for enhancement to occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prereplicative complexes of components of DNA polymerase III holoenzyme of Escherichia coli.

Stepwise reconstitution of the subunits of DNA polymerase III holoenzyme of Escherichia coli offers insights into the organization and function of this multisubunit assembly. A highly processive, holoenzyme-like activity can be generated when the gamma complex, in the presence of ATP and a primed template, activates the beta subunit to form a preinitiation complex, and this is then followed by addition of the core polymerase. Further analysis of early replicative complexes has now revealed: 1) that the gamma complex can stably bind a single-stranded DNA binding protein (SSB)-coated template, 2) that neither SSB coating of the template nor a proper primer terminus is required to form the preinitiation complex, and 3) that the gamma complex stabilizes the preinitiation complex in the presence of ATP and destabilizes it in the presence of adenosine 5'-O-(thiotriphosphate). Based on these findings, a sequence of stages can be formulated for an activation of the beta subunit that enables it to bind the template-primer and thereby interact with the core to create a processive polymerase.     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.