The DNA of Volvox carteri: a biophysical and biosynthetic characterization.

Various biophysical and biosynthetic characteristics of deoxyribonucleic acid (DNA) from Volvox carteri are examined. The DNA from three strains (HK-10,NB-7 and KA-1) is compared, and all strains are shown to contain at least two distinct DNA species which band at densities of 1.714-1.715 and 1.704-1.705 g/cm3 in neutral CsCl and correspond to nuclear and "cytoplasmic" DNA, respectively. Base compositions calculated from these densities, 55-56% G+C for nuclear DNA, and 45-46% G+C for cytoplasmic DNA, are in close agreement with % G+C values estimated from thermal denaturation data. DNA from strain KA-1 has a third component with a buoyant density of 1.693 g/cm3. DNA synthesis is analysed using radioactively labelled heterogeneously grown strains of Volvox carteri and profiles obtained following preparative CsCl density gradient centrifugation are presented. In addition, dissimilarities in patterns of DNA synthesis at various periods in the asexual life cycle are reported for synchronous cultures of strain HK-10. These differences in temporal patterns of DNA synthesis clearly indicate that while nuclear DNA is make to some degree throughout the life cycle, cytoplasmic DNA synthesis appears to occur only at discrete intervals.     

Intranuclear localization of UV-induced DNA repair in human VA13 cells

We have investigated the intranuclear localization of DNA-repair synthesis in G1-phase VA13 human cells. Ultraviolet-irradiated cells were permitted to perform unscheduled DNA synthesis in 3H-thymidine (3H-TdR) and then extracted with nonionic detergent and 2 M NaCl to produce nucleoids in which residual nuclear matrix was surrounded by an extended halo of DNA loops. Autoradiographic analysis of these structures permitted discrimination of DNA repair between the matrix and halo regions. Repair label in nucleoids prepared from cells after exposure to fluences of 2.5-30 J/m2 exhibited a dose-dependent association with the nuclear matrix, which ranged from 80% after 2.5 J/m2 to 50% after 30 J/m2. These results support the view that DNA repair is a nuclear matrix-associated process. This conclusion is in agreement with our preliminary study (Harless et al., 1983) and the results of McCready and Cook (1984) but contrasts with that of Mullenders et al. (1983). Questions concerning the differing experimental designs and their potential effects on the localization of DNA repair are discussed. The implications of these results to previous attempts to isolate chromatin factors associated with DNA repair are also considered.     

Nuclear DNA synthesis is blocked by UV irradiation in Dictyostelium discoideum

We have used a thymidine auxotroph of the simple eukaryote, Dictyostelium discoideum and alkaline sucrose gradients of isolated nuclei to study alterations in DNA synthesis following irradiation of replicating haploid cells with 254 nm UV light. Three responses were characterized using pulse-chase protocols: (1) Lags in DNA synthesis as measured by the amount of label incorporated were 4, 9, and 20 h after 10, 50, and 200 J/m2. (2) The DNA synthesized during a 15-min pulse immediately after irradiation was of lower single strand molecular weight: 7, 3.5, and 3 x 10(6) dalton after 0, 50, and 200 J/m2. (3) The time required for maturation of the nascent DNA to full-sized single strands of about 2 x 10(8) dalton was 45-50 min for unirradiated cells, 3 h after 10 J/m2, and 20 h after 200 J/m2. The DNA of the irradiated cells did not mature uniformly during these delays; instead, a period of no increase in size was followed by a rapid, nearly control rate of maturation. We conclude: (a) at least some UV lesions block elongation of replicons; (b) the elongation of the replicons and their subsequent joining to yield mature high molecular weight DNA occurs after most of the lesions are repaired; (c) the timing of the different aspects of recovery suggest that initiation of replication is also inhibited.     

Regulation of trypanosome DNA glycosylation by a SWI2/SNF2-like protein

Synthesis of the modified thymine base beta-D-glucosyl-hydroxymethyluracil, or J, within telomeric DNA of Trypanosoma brucei correlates with the bloodstream-form-specific epigenetic silencing of telomeric variant surface glycoprotein genes involved in antigenic variation. The mechanism of developmental and telomeric-specific regulation of J synthesis is unknown. We have previously identified a J binding protein (JBP1) involved in propagating J synthesis. We have now identified a homolog of JBP1, JBP2, containing a domain related to the SWI2/SNF2 family of chromatin remodeling proteins that is upregulated in bloodstream form cells and interacts with nuclear chromatin. We show that expression of JBP2 in procyclic form cells leads to de novo J synthesis within telomeric regions of the chromosome and that this activity is inhibited after mutagenesis of conserved residues critical for SWI2/SNF2 function. We propose a model in which chromatin remodeling by JBP2 regulates the initial sites of J synthesis within bloodstream form trypanosome DNA, with further propagation and maintenance of J by JBP1.     

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m2) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m2) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair.     

Modulation of nuclear protein kinase activity and phosphorylation of histone H1 subspecies during the prereplicative phase of rat liver regeneration

We have measured nuclear protein kinase activity during the prereplicative phase of rat liver regeneration. Total nuclear protein kinase activity increased significantly 15-18 h after partial hepatectomy, with the peak of activity occurring at 16 h. DEAE-Sephacel chromatography resolved nuclear protein kinase activity into two cAMP-independent (Ib and II) and two cAMP-dependent (Ia and III) protein kinases. Sixteen h after partial hepatectomy, there was a marked increase in the activities of the nuclear cAMP-dependent protein kinases and a decrease in the activity of nuclear cAMP-independent protein kinase II. Characterization of the two nuclear cAMP-dependent protein kinases revealed them to be identical with the cytosolic type I and II isozymes. Immunotitration of nuclear catalytic subunit and densitometric analysis of autoradiographs from 8-azido-[32P]cAMP-labeled nuclear RI revealed increases in both subunits 16 h afer partial hepatectomy. Concomitantly with the observed increase in nuclear protein kinase activity, we have observed an increase in the phosphorylation of histone H1 subspecies. Administration of the beta-adrenergic antagonist DL-propranolol, which has been shown to cause delays of equal duration in both the second phase of increased intracellular cAMP levels and the initiation of DNA synthesis (MacManus, J. P., Braceland, B. M., Youdale, T., and Whitfield, J. F. (1973) J. Cell. Physiol. 82, 157-164), results in an equivalent delay of increased nuclear protein kinase activity. Colchicine, which has previously been shown to prevent the onset of DNA synthesis (Walker, P. R., and Whitfield, J. F. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 1394-1398), also prevents the increased protein kinase activity normally observed 16 h after partial hepatectomy. We conclude that the onset of DNA synthesis in the regenerating rat liver is preceded by a cAMP-mediated translocation of type I and type II cAMP-dependent protein kinase to the nucleus and phosphorylative modification of histone H1 subspecies. The inhibitory effects of propranolol and colchicine suggest a common cAMP-mediated, colchicine-sensitive link between protein kinase translocation and the initiation of DNA synthesis.     

Suppressive role of endogenous regucalcin in the enhancement of deoxyribonucleic acid synthesis activity in the nucleus of regenerating rat liver

The role of endogenous regucalcin in the regulation of deoxyribonucleic acid (DNA) synthesis activity in the nuclei of regenerating rat liver after partial hepatectomy was investigated. The addition of regucalcin (0.25 and 0.5 microM) in the reaction mixture caused a significant decrease in the nuclear DNA synthesis activity of normal rat liver. This decrease was also seen in the presence of Ca2+-chelator EGTA (0.4 mM), indicating that the effect of regucalcin is not related to nuclear Ca2+. Nuclear DNA activity was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect was completely abolished by the addition of regucalcin (0.5 microM). Nuclear DNA synthesis activity was significantly increased at 24, 48, and 72 h after partial heptectomy. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing nuclear DNA synthesis activity was significantly enhanced at 24 and 48 h after partial hepatectomy. The presence of staurospone (10(-6) M), trifluoperazine (2 x 10(-5) M), or PD98059 (10(-5) M) in the reaction mixture caused a significant decrease in DNA synthesis activity in the nuclei obtained at 24 after partial hepateactomy. The effect of these inhibitors in the presence of anti-regucalcin monoclonal antibody (25 ng/ml) was greater than that in the absence of the antibody. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis activity in regenerating liver with cell proliferation after partial hepatectomy in rats.     

Photoradiation therapy of cutaneous angiosarcomas in mice

Cutaneous angiosarcoma is a relatively rare but devastating malignant vascular tumor. It has a high incidence of recurrence following conventional therapeutic modalities applied either singly or in combination. The increased vascularity of cutaneous angiosarcomas, facilitating selective uptake and retention of a photosensitizing agent, such as hematoporphyrin derivative (HPD), suggests that these tumors would respond well to photoradiation therapy. To study the feasibility of this treatment modality, transplantable hemangiosarcomas were implanted in B6C3F1 female mice. Within 2.5 to 3.5 hours after intraperitoneal administration of HPD, fluorescence was recorded in the tumor as compared with surrounding normal skin. When these photosensitized tumors were exposed to 70 J/cm2 of laser energy from an argon-pumped dye laser at 630 nm, the tumors showed marked necrosis within 24 hours. In another series, the tumors were initially photosensitized with HPD for 3 hours and then treated with laser energy ranging from 0 to 96 J/cm2. A dual labeling procedure demonstrated a dose-related decrease in DNA synthesis rate in tumors that were exposed to 0 to 30 J/cm2 at 24 hours after treatment. Furthermore, tumor tissue exposed to laser energy in excess of 30 J/cm2 showed no significant cellular DNA synthesis. These data, supported by histologic evidence of tissue destruction, suggest that photoradiation therapy has a great potential as a therapeutic modality for cutaneous angiosarcomas.     

Alterations in cell death and cell cycle progression in the UV-irradiated epidermis of bcl-2-deficient mice

The effect of bcl-2 gene ablation on epidermal cell death induced by UV-B irradiation was investigated in mice. Exposure of depilated back skin of bcl-2-/- mice to 0.5 J/cm2 UV-B caused a prolonged increase in the number of epidermal cells showing nuclear DNA fragmentation compared to wild-type littermates. Consistently, skin explants from bcl-2-deficient mice exhibited a higher number of sunburn cells per cm epidermis (16.6+/-2.1 vs 7.0+/-1.5) following exposure to 0.1 J/cm2 UV-B in vitro. Furthermore, UV irradiation failed to increase pre-melanosomes in skin explants from mutant animals, and primary menalocyte cultures derived from bcl-2 null mutants were highly susceptible to UV-induced cell death compared to cultures from wild-type littermates. An accelerated reappearance of proliferating cells, showing nuclear immunoreactivity for Ki-67 and c-Fos, was observed in the UV-irradiated epidermis of bcl-2-deficient mice. Taken together, these findings suggest that effects of UV radiation on epidermal cell death and cell cycle progression are influenced by survival-promoting Bcl-2.     

The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism

DNA damage induced by the carcinogen benzo[a]pyrene dihydrodiol epoxide (BPDE) induces a Chk1-dependent S-phase checkpoint. Here, we have investigated the molecular basis of BPDE-induced S-phase arrest. Chk1-dependent inhibition of DNA synthesis in BPDE-treated cells occurred without detectable changes in Cdc25A levels, Cdk2 activity, or Cdc7/Dbf4 interaction. Overexpression studies showed that Cdc25A, cyclin A/Cdk2, and Cdc7/Dbf4 were not rate-limiting for DNA synthesis when the BPDE-induced S-phase checkpoint was active. To investigate other potential targets of the S-phase checkpoint, we tested the effects of BPDE on the chromatin association of DNA replication factors. The levels of chromatin-associated Cdc45 (but not soluble Cdc45) were reduced concomitantly with BPDE-induced Chk1 activation and inhibition of DNA synthesis. The chromatin association of Mcm7, Mcm10, and proliferating cell nuclear antigen was unaffected by BPDE treatment. However, the association between Mcm7 and Cdc45 in the chromatin fraction was inhibited in BPDE-treated cells. Chromatin immunoprecipitation analyses demonstrated reduced association of Cdc45 with the beta-globin origin of replication in BPDE-treated cells. The inhibitory effects of BPDE on DNA synthesis, Cdc45/Mcm7 associations, and interactions between Cdc45 and the beta-globin locus were abrogated by the Chk1 inhibitor UCN-01. Taken together, our results show that the association between Cdc45 and Mcm7 at origins of replication is negatively regulated by Chk1 in a Cdk2-independent manner. Therefore, Cdc45 is likely to be an important target of the Chk1-mediated S-phase checkpoint.     

Effect of mutation of amino acids 246-251 (KRKHKK) in HSP72 on protein synthesis and recovery from hypoxic injury

Heat shock protein (HSP)72, the inducible form of HSP70, protects cells against a variety of injuries, but underlying mechanisms are poorly defined. To investigate the protective effects of HSP72, multiple clones expressing wild-type (WT) HSP72 and two mutants with defective nucleolar and nuclear localization (M45 and 985A, respectively) were made with the tet-off system in C2C12 cells. Four different parameters of cell function/injury were examined after simulated ischemia: protein synthesis, polysome formation, DNA synthesis, and lactate dehydrogenase (LDH release). Overexpression of WT HSP72 was also compared to nontransfected C2C12 cells. As expected, overexpression of HSP72 protected against simulated ischemia and reoxygenation for all parameters. In contrast, both M45 and 985A showed abnormal protein synthesis and polysome formation, both after simulated ischemia and under control conditions. Total RNA was slightly reduced in M45 and 985A at baseline, but 1 h after hypoxia, RNA levels were protected in all clones but significantly decreased in nontransfected C2C12 cells. Clones expressing 985A had nuclear retention of mRNA, suggesting that HSP72 is needed for nuclear export of RNA. All clones, both WT and mutant, had protection of DNA synthesis compared to C2C12 cells, but 985A had greater release of LDH after injury than any other group. These results support a multifactoral protective effect of HSP72, some aspects dependent on nuclear localization with stress and some not. The protection of protein synthesis and polysome formation, and abnormalities in these with the mutants, support a role for HSP72 in these processes both in the normal cell and in injury.     

Mechanisms of photochemotherapy-induced apoptotic cell death in lymphoid cells.

Previous studies we performed showed that 8-methoxypsoralen in combination with ultraviolet A light (photochemotherapy) caused DNA damage and that this caused nucleotide depletion in peripheral blood leukocytes, secondary to an active form of programmed cell death, poly(ADP-ribosyl)ation. Further studies revealed that 24 h after exposure to 10 J/cm2 ultraviolet A light and 8-methoxypsoralen (300 ng/mL), apoptotic cells increased from 3 (control) to 31% (p less than 0.001). Ultraviolet A light alone also significantly increased the number of apoptotic cells. These morphological changes were confirmed by parallel findings on DNA electrophoresis. Treatment with 2 to 5 J/cm2 of ultraviolet A light and 8-methoxypsoralen caused an approximately 30% increase in cytosolic free calcium levels in peripheral blood leukocytes 1 h after exposure. Associated with this was a 51% increase in 45Ca2+ uptake over the first 60 min. Similar findings in a different lymphoid cell (CCRF-CEM) confirmed the results obtained with peripheral blood leukocytes. The use of calcium-free medium prevented a rise in cytosolic free calcium and decreased the number of cells undergoing apoptotic cell death. Cycloheximide inhibited ultraviolet A light - 8-methoxypsoralen induced apoptosis in CCRF-CEM cells; it also decreased calcium levels in control CCRF-CEM cells. This study shows that ultraviolet A light - 8-methoxypsoralen caused apoptotic cell death in lymphoid cells; this appeared to be associated with calcium influx, presumably because of the requirement of endogenous endonucleases for calcium.     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Deoxyribonucleic Acid Synthesis in Saccharomyces cerevisiae Cells Permeabilized with Ether

Cells of Saccharomyces cerevisiae permeabilized by treatment with ether take up and incorporate exogenous deoxynucleoside triphosphate into deoxyribonucleic acid (DNA). With rho(+) strains, more than 95% of the product was mitochondrial DNA (mtDNA). This report characterizes ether-permeabilized yeast cells and describes studies on the mechanism of mtDNA synthesis with this system. The initial rate of in vitro mtDNA synthesis with one strain (X2180-1Brho(+)) was close to the rate of mtDNA replication in vivo. The extent of synthesis after 45 min was sufficient for the duplication of about 25% of the total mtDNA in the cells. The incorporated radioactivity resulting from in vitro DNA synthesis appeared in fragments that were an average of 30% mitochondrial genome size. Density-labeling experiments showed that continuous strands of at least 7 kilobases after denaturation, and up to 25 kilobase pairs before denaturation, were synthesized by this system. Pulse-chase experiments demonstrated that a large proportion of DNA product after short labeling times appeared in 0.25-kilobase fragments (after denaturation), which served as precursors of high-molecular-weight DNA. It is not yet clear whether the short pieces participate in a mechanism of discontinuous replication similar to that of bacterial and animal cell chromosomal DNA or whether they are related to the rapidly turning over, short initiation sequence of animal cell mtDNA. In rho(0) strains, which lack mtDNA, the initial rate of nuclear DNA synthesis in vitro was 1 to 2% of the average in vivo rate. With temperature-sensitive DNA replication mutants (cdc8), the synthesis of nuclear DNA was temperature sensitive in vitro as well, and in vitro DNA synthesis was blocked in an initiation mutant (cdc7) that was shifted to the restrictive temperature before the ether treatment.     

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium

When resting WI-38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine-³H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl-soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non-proliferating cells were stable for at least 12 hours. 2-mercapto-1-(β-4-pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI-38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.     

Suppressive effect of endogenous regucalcin on deoxyribonuclic acid synthesis in the nuclei of rat renal cortex

The effect of regucalcin, a regulatory protein of Ca2+ signaling, on deoxyribonucleic acid (DNA) synthesis activity in the nuclei isolated from rat renal cortex was investigated. The addition of calcium chloride (10-100 microM) in the reaction mixture containing the nuclei caused a significant decrease in DNA synthesis activity. Nuclear DNA synthesis activity was significantly raised in the presence of EGTA (1 mM), a chelator of Ca2+, indicating that nuclear Ca2+ has an inhibitory effect. Regucalcin (0.1-0.5 microM) added in the reaction mixture in the presence of either EGTA (1 mM) or calcium chloride (50 microM) had a significant inhibitory effect on nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity. This increase was completely abolished by the addition of regucalcin (0.5 microM). The effect of anti-regucalcin monoclonal antibody in increasing DNA synthesis was enhanced in the presence of EGTA. Additionally, an inhibitory effect of calcium chloride (10 or 50 microM) was enhanced in the presence of anti-regucalcin monoclonal antibody (25 ng/ml). The present study demonstrates that endogenous regucalcin has a suppressive effect on DNA synthesis in the nuclei of rat renal cortex.     

Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation

In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the subsequent binding of polymerase to the loaded clamp. Recently published evidence suggests that holoenzyme assembly in the T4 replisome may take place via more than one pathway [Zhuang, Z., Berdis, A. J., and Benkovic, S. J. (2006) Biochemistry 45, 7976-7989]. To demonstrate unequivocally whether there are multiple pathways leading to the formation of a productive holoenzyme, single-molecule fluorescence microscopy has been used to study the potential clamp loading and holoenzyme assembly pathways on a single-molecule DNA substrate. The results obtained reveal four pathways that foster the formation of a functional holoenzyme on DNA: (1) clamp loader-clamp complex binding to DNA followed by polymerase, (2) clamp loader binding to DNA followed by clamp and then polymerase, (3) clamp binding to DNA followed by clamp loader and then polymerase, and (4) polymerase binding to DNA followed by the clamp loader-clamp complex. In all cases, MgATP is required. The possible physiological significance of the various assembly pathways is discussed in the context of replication initiation and lagging strand synthesis during various stages of T4 phage replication.     

Differential chromosomal and mitochondrial DNA synthesis in temperature-sensitive mutants ofUstilago maydis

Summary The amount and type of residual DNA synthesis was determined in eight temperature-sensitive mutants of the smut fungusUstilago maydis after incubation at the restrictive temperature (32° C) for eight hours. Mutantsts-220,ts-207,ts-432 andts-346 were found to have an overall reduction in the synthesis of both nuclear and mitochondrial DNA in comparison to the wild-type. In mutantsts-20,tsd 1-1,ts-84 andpol 1-1 nuclear DNA synthesis was depressed relative to mitochondrial synthesis. The DNA-polymerase mutantpol 1-1 had persistent nuclear synthesis at about 50% of the rate of synthesis of mitochondrial DNA and similar behavior was observed in a diploid homozygous strain. Mutantts-84 had an initial burst of DNA synthesis which was reduced for nuclear but not mitochondrial synthesis after three hours preincubation at 32°C.tsd 1-1 andts-20 had nuclear residual synthesis amounting to about 25% of the relative rate of mitochondrial synthesis with correlates to increasing UV sensitivity of these strains on incubation at 32° C. Apol 1-1ts-84 double mutant had an additive loss of nuclear DNA synthesis which indicates that the steps of replication involved may be sequential.     

A Bacteriophage of Bacillus subtilis Which Forms Plaques Only at Temperatures Above 50 C III. Inhibition of TSP-1-Specific Deoxyribonucleic Acid Synthesis at 37 C and 45 C

The effect of temperature on phage-specific deoxyribonucleic acid (DNA) synthesis was studied in TSP-1-infected Bacillus subtilis. This was facilitated by selectively inhibiting host DNA synthesis with 6-(p-hydroxyphenylazo)-uracil. The results indicated that TSP-1 DNA synthesis did not continue at 37 C and was immediately shut down after transfer to this temperature. Incubation at 45 C greatly reduced TSP-1 DNA synthesis. Phage-specific DNA synthesis could resume at 53 C, however, when the infected culture was returned to 53 C after a 2-min incubation period at 37 C. The results suggest that the inhibition of phage DNA synthesis at 37 C is reversible. Since infected cultures returned to 53 C after 2 min at 37 C could not complete the replicative cycle, the irreversible inhibition of yet another intermediate step was suggested.