DNA Chain Elongation Research Articles

Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli.

msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M. (1989) Science 243, 1033-1038). The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT). These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator. msDNA-Ec67 was produced only when cells were transformed with both plasmids. In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT. Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound. This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue. The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA. DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder. The DNA sequence determined from this ladder completely agreed with the msDNA sequence. The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase. This clearly demonstrates that RNA but not DNA is responsible for the msDNA production. A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model. In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT. Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R. A., and Yee, T. (1991) J. Biol. Chem. 266, 14497-14503).

Effect of UVC Light on Growth, Incorporation of Thymidine, and DNA Chain Elongation in Cells Derived from the Indian Meal Moth and the Cabbage Looper

After exposure to 10 or 20 J/m2 UVC light, cells of the UMN-PIE-1181 line, an embryonic cell line derived from the Indian meal moth, Plodia interpunctella, exhibited a rapid and prolonged depression in the rate of incorporation of [3H]thymidine, whereas cells of the TN-368 line, an ovarian cell line derived from Trichoplusia ni, the cabbage looper, showed only a slight drop in incorporation and a rapid recovery after exposure to 10 or 40 J/m2 UVC light. The extent of this depression was not correlated to the amount of cell killing by UVC light in these cell lines or in IAL-PID2 cells. Blockage of fork progression was correlated to the depression in thymidine incorporation. TN-368 cells exhibited little blockage after exposure to 10 or 20 J/m2 UVC light, whereas UMN-PIE-1181 cells exhibited significant blockage at these fluences. Photoreactivation did not entirely relieve blockage, depression in thymidine incorporation, or cell killing, indicating that, although the (5-6) dimer appears to be the major lesion responsible for these effects, other lesions such as the (6-4) photoproduct may play a role.

Effect of arsenite on the DNA repair of UV-irradiated Chinese hamster ovary cells.

Arsenite, an ubiquitous human carcinogen, has been shown to enhance the cytotoxicity, mutagenicity and clastogenicity of UV light in mammalian cells. Arsenite may exert its co-genotoxic effects by inhibiting DNA repair. Results from alkaline sucrose gradient sedimentation show that arsenite did not accumulate UV-induced DNA strand breaks in Chinese hamster ovary (CHO) K1 cells as aphidicolin plus hydroxyurea (HU) did. These data indicate that arsenite did not inhibit the activity of DNA polymerase alpha in UV repair. Treatment with arsenite before UV irradiation slightly reduced the DNA strand breaks accumulated by cytosine beta-D-arabinofuranoside (AraC) plus HU. This effect implies that arsenite only slightly inhibited the incision of UV-induced DNA adducts. The low molecular weight DNA accumulated by post-UV incubation with AraC plus HU shifted to high molecular weight upon the incubation of cells in drug-free medium, but this shifting was prohibited by the presence of arsenite. This suggests that arsenite inhibited the rejoining of DNA strand breaks. When a pulse-chase labelling procedure was applied on UV-irradiated cells, the chain elongation of nascent DNA was strongly inhibited by post-incubation with arsenite. These data show that arsenite inhibited post-replication repair in UV-irradiated cells. Therefore, the steps inhibited by arsenite in UV-induced DNA repair in CHO K1 cells are different from human fibroblasts in which the inhibition of excision of pyrimidine dimers by arsenite was reported to be the major target.

Blockage of polymerase-catalyzed DNA chain elongation by chemically modified cytosine residues in templates and the release of blockage for readthrough.

The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5,6-dihydrocytosine-6-sulfonate residues. We have studied the mechanism of the blockage, using as templates bisulfite-hydrazine (and -methoxyamine)- modified single strand phage-M13mp2 DNA and synthetic oligonucleotides. Both N4-amino-5,6-dihydrocytosine-6-sulfonate and N4-methoxy-5,6-dihydrocytosine-6-sulfonate residues blocked the elongation at one nucleotide before these sites. In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites. It seems possible that conformational distortion of the template near these sites is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted. In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites. Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site. These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions. This observation suggests that these sites can be read by E. coli DNA polymerases in vivo with accompanying errors.

Intranuclear accumulation of subgenomic noninfectious human cytomegalovirus DNA in infected cells in the presence of ganciclovir.

In preparation for an attempt to elucidate some aspects of the interaction between ganciclovir and human cytomegalovirus (HCMV) DNA replication in cells infected with HCMV, we developed a dot blot DNA-DNA hybridization technique to quantify intracellular HCMV DNA replication. We studied the effect of ganciclovir on the time course of HCMV DNA replication in human fibroblasts. Ganciclovir resulted in complete cessation of the production of infectious virus, as detected by the plaque assay. However, viral DNA synthesis, as measured by dot blot DNA-DNA hybridization with cloned HCMV DNA BamHI C fragment probe, continued in the presence of ganciclovir at 10 times the 50% effective dose (i.e., 10 micrograms/ml). The continuation of viral DNA synthesis in ganciclovir-treated cultures leads to the intranuclear accumulation of short (subgenomic) HCMV DNA fragments. These DNA fragments are neither packaged nor released into the culture medium. Furthermore, the short DNA fragments were detected only by the BamHI C probe from the center of the unique long segment of the HCMV genome. The failure of the DNA probes from the termini of HCMV genome (BamHI-Q and HindIII-M) to detect the short DNA fragments and the intranuclear localization of these fragments suggest that these short fragments may lack the signal sequences necessary for packaging and release as infectious virions. These data strongly suggest that the anti-HCMV activity of ganciclovir is due mainly to the prevention of viral DNA chain elongation which results in the intranuclear accumulation of incomplete noninfectious viral DNA fragments.

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.

A DNA replication gene maps near terC in Escherichia coli K12

Temperature-sensitive mutants that filamented at the non-permissive temperature were isolated by specific mutagenesis of the terminus region of the Escherichia coli chromosome. Two of them, mapping at about 35 min, failed to divide due to inhibition of DNA replication. Further characterization indicated that these mutants are temperature-sensitive for DNA chain elongation.

Role of DNA replication in carrier-ligand-specific resistance to platinum compounds in L1210 cells

L1210 cell lines have been described that are sensitive to most platinum compounds (L1210/0), resistant to ethylenediamine (en)-Pt but sensitive to diaminocyclohexane (dach)-Pt (L1210/DDP), and resistant to dach-Pt but sensitive to en-Pt (L1210/DACH). We have examined the effect of the dach and en carrier ligands on the ability of Pt-DNA adducts to inhibit DNA replication. Alkaline sucrose gradient sedimentation was used to determine the influence of both carrier ligands on the inhibition of replicon initiation and DNA chain elongation. Initiation of replicons was inhibited by Pt-DNA adducts to a greater extent than chain elongation in all three cell lines. Inhibition of replicon initiation was affected by the nature of the platinum carrier ligands only in the L1210/DACH cells in which 7.8-fold more dach-Pt adducts than en-Pt adducts were required to reach 63% inhibition. However, a strong carrier ligand effect was observed on the inhibition of DNA chain elongation in both the L1210/DDP and L1210/DACH cell lines. The L1210/DDP cell line required 4-fold more en-Pt adducts than dach-Pt adducts to inhibit DNA chain elongation by 63%. In the L1210/DACH cell line, 2.7-fold more dach-Pt adducts than en-Pt adducts were required for 63% inhibition. The L1210/0 cell line demonstrated no carrier ligand specificity for inhibition of chain elongation. Significant replicative bypass of Pt-DNA adducts was observed even in the L1210/0 cell line in that greater than 50 Pt-DNA adducts per 100 kb were required for 63% inhibition. The same level of inhibition was reached with 1.25 adducts of benzo[a]pyrene diolepoxide I per 100 kb. These data suggest that L1210 cells are capable of substantial replicative bypass of Pt-DNA adducts. Furthermore, the bypass of Pt-DNA adducts is increased in resistant L1210 cells and is markedly dependent on the nature of the platinum carrier ligand.

Etoposide Sensitivity and Topoisomerase II Activity in Chinese Hamster V79 Monolayers and Small Spheroids

Chinese hamster V79 cells grown in suspension culture as spheroids are more resistant than monolayers to killing and mutation by ionizing radiation. A change in DNA conformation appears to accompany this increase in radiation resistance. We were therefore interested in whether the activity of topoisomerase II, a nuclear enzyme involved in DNA conformational changes and possibly in DNA repair, might differ in monolayers and small spheroids. One-day-old spheroid cells were more resistant than monolayer cells to the toxic effects of etoposide, a topoisomerase II inhibitor. Fewer strand breaks were induced by etoposide in spheroid DNA than monolayer DNA, as measured by the DNA precipitation and alkali unwinding assays, although identical amounts of damage were produced in monolayers and spheroids by the topoisomerase I inhibitor camptothecin, and the cell cycle specific agent, 5-fluorouracil. There was no evidence of a subpopulation of spheroid cells which were more resistant to etoposide, and no change in the rate of incorporation or DNA chain elongation in spheroids compared to monolayers. Topoisomerase II activity in 1-day-old spheroids, measured by decatenation of trypanosome kinetoplast DNA, was reduced to 68% of the monolayer value; in 3-4-day-old spheroids the level was 32.5%. These results indicate that topoisomerase II activity and sensitivity to a topoisomerase II inhibitor are reduced in 1-day-old spheroid cells. We suggest that the decrease in the activity of this enzyme may be linked to the change in DNA conformation in spheroids and the decrease in their radiation sensitivity.

Cloning and cell cycle-dependent expression of DNA replication gene dnaC from Caulobacter crescentus

Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence corresponded to dnaC, we isolated the gene by genetic complementation of the temperature-sensitive allele dnaC303 on DNA fragment that contained a Tn5 insertion element tightly linked by transduction to dnaC. The size of the dnaC gene was estimated to be 1,500 bp or less based on the pattern of complementation by subcloned restriction and BAL 31 deletion fragments. Nuclease S1 assays were used to map the transcription start site and to determine the pattern of dnaC expression in the cell cycle. Large amounts of the dnaC transcript began to accumulate only in the late G1 period of the swarmer cell and then peaked early during chromosome replication. We confirmed that the gene is periodically transcribed by monitoring the rate of beta-galactosidase synthesis directed by a dnaC promoter-lacZ fusion in a synchronous cell culture. dnaC is the first C. crescentus cell cycle gene whose regulation has been reported, and the discontinuous pattern of its expression suggests that the DNA synthetic period in these dimorphic bacteria is regulated in part by the stage-specific expression of DNA replication genes.

Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells

Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of DNA replication as well as a temporary blockage of DNA fork progression. Recently there has been considerable debate as to whether the (5–6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cells lines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of DNA fork progression. Specifically, after exposure to 2.5 J/m2, AA8 cells recover normal rates of DNA replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of DNA fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of DNA fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of DNA fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.

Inhibition of DNA synthesis and cytotoxic effects of some DNA topoisomerase II and gyrase inhibitors in Chinese hamster V79 cells

In this study, some DNA topoisomerase II and gyrase inhibitors have been identified as inhibitors of polymerization of deoxyribonucleotides [novobiocin (NVB), nalidixic acid (NDA), oxolinic acid (OXA)], or inhibitors of replicon initiation and DNA-chain elongation [etoposide (VP-16), teniposide (VM-26), 4′-(9-acridinylamino)methansulfon- m-anisidine ( m-AMSA), ellipticine (ELT)]. The inhibitors of deoxyribonucleotide polymerization produced a significant (> 85%) suppression of [ 3H]thymidine incorporation into V79 cells within 20 min of treatment, followed by a rapid recovery of DNA synthesis, and reduced cell killing. In contrast, the inhibitors of replicon initiation and DNA-chain elongation needed about 60 min to induce a partial, but irreversible inhibition of DNA replication, associated with extensive cell killing.

Synergy of inhibition of DNA synthesis in human bone marrow by azidothymidine plus deficiency of folate and/or vitamin B12?

The effect of azidothymidine (Zidovudine, AZT) on pyrimidine (thymidine, deoxyuridine, and thymidine triphosphate) incorporation into DNA in folate- and/or vitamin B12-deficient and normal human bone marrow cells was studied to investigate whether such vitamin deficiency affects susceptibility to AZT-induced hematologic toxicity. Bone marrow cells from 12 patients were studied: 5 had folate and/or vitamin B12 deficiency; 7 controls included 5 with anemia related to chronic disease and 2 with iron deficiency. At 0.2 microM AZT (3 hr, 37 degrees C), the approximate pharmacologic serum trough level, pyrimidine incorporation into DNA was suppressed by 12 to 19% in folate- and/or vitamin B12-deficient cells and by 16 to 23% in normal cells. At 2.0 microM AZT (3 hr, 37 degrees C), the approximate pharmacologic serum peak level, this was suppressed by 15 to 40% in folate- and/or vitamin B12-deficient cells and by 32 to 47% in controls. Deoxyuridine incorporation into DNA was inhibited significantly greater than thymidine at 2.0 microM AZT (3 hr, 37 degrees C) in both groups. Inhibition of deoxyuridine incorporation was not reversed with methyltetrahydrofolate or vitamin B12. There tended to be less striking suppression by AZT of deoxyuridine incorporation into DNA in bone marrow cells from vitamin B12-deficient patients, which was made more striking by adding vitamin B12. This suggests that some of what passes for "AZT damage" to bone marrow cells may in fact be coincident deficiency of vitamin B12. AZT inhibition of DNA synthesis in 3 hr bone marrow cultures is relatively consistent in a variety of hematologic disorders. As approximately two-thirds of AIDS patients appear to be in negative balance with respect to folate and/or vitamin B12, the fact that AZT-induced inhibition of pyrimidine incorporation into DNA is occurring in cells which may be megaloblastic, i.e., in a state of impaired DNA synthesis, suggests that these cells may be more susceptible to AZT toxicity. The data also support the notion that AZT inhibition results predominantly from termination of DNA chain elongation. Whether folate or vitamin B12 supplementation may partially overcome apparent "AZT inhibition" of DNA synthesis (hematologic toxicity) and whether the benefit of such therapy exceeds the risk will require further study.

DNA replication in relation to S phase duration in six angiosperm annual species.

Using fiber autoradiography, replicon sizes and DNA replication fork rates were studied in 6 diploid angiosperm annual species with different 2C DNA values and S phase durations (Ts). The only correlations were found between Ts and Rs/Ts ratios (positive correlation, p=0.001) and between 2C DNA content and the rate of fork movement (negative correlation, p=0.05). The observations raise the possibility of a connection between the mechanisms involved in DNA chain elongation and the amount of repetitive sequences and between Ts and the functional arrangements of replicons.

Inhibition of Chinese hamster ovary cell DNA synthesis by hydrogen peroxide

The DNA synthesis inhibitory effect of hydrogen peroxide has been examined under a number of experimental conditions. Results have indicated that the effect of the oxidant is more pronounced when the treatment is performed at 37°C than at 4°C and in low density as compared to high density cultures. In addition, similar levels of inhibition were achieved by measuring the incorporation of radiolabelled thymidine in the presence of, or following treatment with, the oxidant. Although early events seem to be responsible for the decreased rate of DNA synthesis, it would appear that hydrogen peroxide does not alter thymidine extracellularly and/or decrease the transport of the nucleoside across the plasma membrane, which may actually be slightly augmented. Thus, the previously illustrated results may represent an underestimate of the actual capacity of the oxidant to reduce DNA synthesis. This inference is further supported by the fact that the effect of hydrogen peroxide appears markedly enhanced in cells preloaded with the radiolabelled precursor. A temporal relationship seems to exist between the steady state level of DNA single strand breaks and the extent of DNA synthesis inhibition by hydrogen peroxide. The oxidant has no effect on DNA chain elongation. In conclusion, data presented in this paper suggest that early events, involving selective effects on replicon initiation, mediate the DNA synthesis inhibitory effect of hydrogen peroxide.

Isolation of temperature-sensitive cell cycle mutants from mouse FM3A cells. Characterization of mutants with special reference to DNA replication.

A large number of mutants that are temperature sensitive (ts) for growth have been isolated from mouse mammary carcinoma FM3A cells by an improved selection method consisting of cell synchronization and short exposures to restrictive temperature. The improved method increased the efficiency of isolating DNA ts mutants, which showed a rapid decrease in DNA-synthesizing ability after temperature shift-up. Sixteen mutants isolated by this and other methods were selected for this study. Flow microfluorometric analysis of these mutants cultured at a nonpermissive temperature (39 degrees C) for 16 h indicated that five clones were arrested in the G1 to S phase of the cell cycle, six clones were in the S to G2 phase, and two clones were arrested in the G2 phase. The remaining three clones exhibited 8C DNA content after incubation at 39 degrees C for 28 h, indicating defects in mitosis or cytokinesis. These mutants were classified into 11 complementation groups. All the mutants except for those arrested in the G2 phase and those exhibiting defects in mitosis or cytokinesis showed a rapid decrease in DNA synthesis after temperature shift-up without a decrease in RNA and protein synthesis. The polyomavirus DNA cell-free replication system, which consists of polyomavirus large tumor antigen and mouse cell extracts, was used for further characterization of these DNA ts mutants. Among these ts mutants, only the tsFT20 strain, which contains heat-labile DNA polymerase alpha, was unable to support the polyomavirus DNA replication. Analysis by DNA fiber autoradiography revealed that DNA chain elongation rates of these DNA ts mutants were not changed and that the initiation of DNA replication at the origin of replicons was impaired in the mutant cells.

DNA primase-DNA polymerase α assembly from mouse FM3A cells: Purification of constituting enzymes, reconstitution, and analysis of RNA priming as coupled to DNA synthesis

The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.

Heat Shock Protein-mediated Disassembly of Nucleoprotein Structures Is Required for the Initiation of Bacteriophage λ DNA Replication

Abstract Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo. We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high. GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level. We demonstrate in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda). Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E. coli DnaB helicase. With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template. Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation. We infer from our results that DnaK and DnaJ function in normal E. coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions. We also provide evidence that neither the lambda O and P initiators nor the E. coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.

Cloning and sequence analysis of the Saccharomyces cerevisiae RAD9 gene and further evidence that its product is required for cell cycle arrest induced by DNA damage.

Procaryotic and eucaryotic cells possess mechanisms for arresting cell division in response to DNA damage. Eucaryotic cells arrest division in the G2 stage of the cell cycle, and various observations suggest that this arrest is necessary to ensure the completion of repair of damaged DNA before the entry of cells into mitosis. Here, we provide evidence that the Saccharomyces cerevisiae RAD9 gene, mutations of which confer sensitivity to DNA-damaging agents, is necessary for the cell cycle arrest phenomenon. Our studies with the rad9 delta mutation show that RAD9 plays a role in the cell cycle arrest of methyl methanesulfonate-treated cells and is absolutely required for the cell cycle arrest in the temperature-sensitive cdc9 mutant, which is defective in DNA ligase. At the restrictive temperature, cell cycle progression of cdc9 cells is blocked sometime after the DNA chain elongation step, whereas cdc9 rad9 delta cells do not arrest at this point and undergo one or two additional divisions. Upon transfer from the restrictive to the permissive temperature, a larger proportion of the cdc9 cells than of the cdc9 rad9 delta cells forms viable colonies, indicating that RAD9-mediated cell cycle arrest allows for proper ligation of DNA breaks before the entry of cells into mitosis. The rad9 delta mutation does not affect the frequency of spontaneous or UV-induced mutation and recombination, suggesting that RAD9 is not directly involved in mutagenic or recombinational repair processes. The RAD9 gene encodes a transcript of approximately 4.2 kilobases and a protein of 1,309 amino acids of Mr 148,412. We suggest that RAD9 may be involved in regulating the expression of genes required for the transition from G2 to mitosis.