DNA replication in Physarum polycephalum: characterization of replication products in vivo.

Synchronous plasmodia of Physarum polycephalum in DNA synthesis were pulse-labelled with [oH]- thymidine for time periods of 15 seconds up to 9 minutes, or given a 30 seconds pulse followed by chase periods of 9 minutes up to 6 hours. Sedimentation analysis in alkaline sucrose gradients revealed at least five species of single stranded DNA14 molecules in the pulse experiments. Co-sedimentation of [14C]-labelled phage-DNA gave relative S-values of 5-7, 13-15, 23-25, 30 and 33-35 for these DNA molecules, all of which can be chased into DNA of higher molecular weight.     

DNA replication in Physarum polycephalum: characterization of DNA replication products made in vivo in the presence of cycloheximide in strains sensitive and resistant to cycloheximide.

Synchronous plasmodia of cycloheximide-sensitive and cycloheximide-resistant strains of Physarum polycephalum were labelled with 3[H]-deoxyadenosine in pulse and pulse-chase experiments in presence and absence of cycloheximide. The replication products were studied with alkaline sucrose gradient sedimentation analysis. We show that the action of cycloheximide on DNA replication in Physarum is mediated through the ribosome, since the ribosomally located resistance also makes the plasmodial DNA replication refractile to the action of cycloheximide. Cycloheximide caused inhibition of three stages in DNA replication in the wild type: first, the formation of primary replication units ("Okazaki" size fragments), secondly, the ligation of primary units into secondary ("Replicon" size) units and thirdly, the ligation of secondary units into mature DNA.     

DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum

Nuclei were isolated from synchronized plasmodia of a true slime mold, Physarum polycephalum, in S-phase, and DNA synthesis in the nuclei was studied in vitro. The nuclei catalyzed DNA synthesis at the rate of 0.7 ng DNA/1.0 X 10(6) nuclei/30 min at 25 degrees C, which was 5 times higher than that catalyzed in G2-phase nuclei. The DNA synthesis required Mg2+, four kinds of deoxyribonucleoside 5'-triphosphates and ATP, suggesting that the mode of synthesis is a replicative-type, but not a repair-one. Sedimentation analysis of the DNA products revealed that the nuclei produced 2-4S DNA fragments mainly during a 30-sec pulse incubation, and 2-4S, 5-12S and longer fragments during a 15-min incubation. The pulse- and chase-labeling experiments showed that the 2-4S fragments shifted discontinuously to longer fragments. These results indicate that the nuclei catalyze the formation of 2-4S Okazaki fragments first and then their subsequent ligation. Eighty % and 96% of the DNA synthesis was inhibited by 200 micrograms/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 microM 2',3'-dideoxythymidine 5'-triphosphate. These results suggest that the DNA synthesis is catalyzed by the alpha-type DNA polymerase of Physarum polycephalum.     

DNA replication in homogenates of Physarum polycephalum

Homogenates of Physarum polycephalum incorporate [³H] dATP into nuclear DNA at an initial rate of approximately 15% of the in vivo rate. To attain this level of synthesis, cultures are homogenized in a medium containing Mg⁺⁺, EGTA, glucose and spermine. Incorporation is strongly stimulated by the addition of ATP and all four deoxyribonucleoside triphosphates to homogenates prior to incubation. Various inorganic cations other than Mg⁺⁺ either do not affect synthesis or are inhibitory. Incorporation is inhibited by a nonionic detergent, Triton X-100. DNA synthesis in this cell-free nuclear system is similar in several respects to that which occurs in vivo: (1) The rate of DNA synthesis in the intact organism at a given time in the mitotic cycle is reflected by the level of synthesis in homogenates prepared from cultures at that time of the cycle; (2) DNA strands labeled in vitro exhibit alkaline sucrose density gradient sedimentation properties similar to those of daughterstrand DNA pulse-labeled in vivo; and (3) Homogenates of cultures which were pre-treated with cycloheximide incorporate [³H]dATP at about 60% of the level observed in homogenates of untreated controls.     

ADP-ribosylation in isolated nuclei of Physarum polycephalum.

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.     

DNA replication in Physarum polycephalum: bidirectional replication of DNA within replicons.

The direction of replication of DNA within replicons of Physarum polycephalum was studied by pulse-labelling with 5-bromouracil-deoxyriboside (BrdUrd) and 3H-adenosine deoxyriboside (dAdo), followed by ultraviolet- (UV) -photolysis and analysis of molecular weights of single strand DNA fragments on alkaline sucrose gradients. Newly made DNA within replicons at all stages of completion is split in two equal halves upon UV irradiation when BrdUrd was given at the time of initiation of DNA synthesis. This shows that replication within replicons of Physarum polycephalum starts at an origin located in the center of each unit, proceeding bidirectionally from this origin.     

Isolation and DNA content of nuclei of Physarum polycephalum

Methods have been developed for isolation of nuclei from Physarum polycephalum at various stages of the life cycle and mitotic figures and nucleoli from the plasmodial stage. Organelles of growing plasmodia and myxamoebae were isolated by Waring blending in 0.25 M sucrose, 0.1% Triton X-100, 0.01 M CaCl2 (0.001 M for nucleoli) and 0.01 M Tris buffer, pH 7.2, and centrifuging through 1 M sucrose. The same procedure was used for starving cultures, except that before homogenization starving and sporulating plasmodia were washed with 0.01 M EDTA in 0.25 M sucrose, and spherules were washed with EDTA-sucrose and broken in the French pressure cell.     

Studies on the mechanism of DNA replication in Physarum polycephalum

The synthesis of single-stranded DNA subunits (4 × 10⁷ daltons) in Physarum polycephalum was studied by alkaline sucrose density gradient centrifugation. The results were compared with the synthesis of the double-stranded DNA molecules (2.3 × 10⁸ daltons) which they comprise, as determined from neutral sucrose density gradient centrifugation patterns. Although the initiation of synthesis of most double-stranded DNA molecules takes place relatively early in the S period, synthesis of the subunits within them is initiated throughout at least the first two hours of this period. Similarly, replicating (presumably forked) DNA molecules appear to split into daughter DNA molecules prior to the completion of synthesis of the subunits therein. The average rate of DNA chain elongation within subunits is 0.3 × 10⁶ daltons/minute. It is suggested that alkaline sucrose density gradient centrifugation may be a more sensitive method for determining the time required for the completion of replication than other methods based solely on the incorporation of radioactive DNA precursors into an acid-insoluble product.     

Regulation of DNA replication in the nuclei of the slime mold Physarum polycephalum. Transplantation of nuclei by plasmodial coalescence

Nuclei in G2 phase of the slime mold Physarum polycephalum, when transplanted, by plasmodial coalescence, into an S-phase plasmodium, failed to start another round of DNA synthesis. In the reciprocal combination, S-phase nuclei in a G2-phase host continued DNA synthesis for several hours without appreciable decrease in rate. It is suggested that the beginning of DNA replication is determined by an event, either during or shortly after mitosis, which renders the chromosomes structurally competent for DNA replication.     

Structure of a beta-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated beta-D-galactan ([alpha]D+8degrees) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 leads to 4)-, but also of (1 leads to 3)-, and (1 leads to 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extracellular polysaccharide produced by P. polycephalum.     

ADP-ribosyltransferase in isolated nuclei during the cell cycle of Physarum polycephalum.

ADP-ribosyltransferase was measured in isolated nuclei of Physarum polycephalum. Activity was determined with and without exogenous DNA and histones. During the synchronous cell cycle the activity measured with exogenous substrates exhibited a typical peak enzyme pattern with a maximum of activity in S-phase, whereas activity measured without exogenous substrates displayed a step enzyme pattern. Both activities doubled in each cell cycle.     

DNA replication in Physarum polycephalum: UV photolysis of maturing 5-bromo-deoxyuridine substituted DNA.

Combinations of 5-bromodeoxyuridine (BrdUrd) and 3H-deoxyadenosine (3H-DAdo) short pulses were given in the synchronous DNA-replication period of Physarum polycephalum. After a chase period, UV-photolysis products were analyzed on alkaline sucrose gradients. This strategy has allowed the following conclusions. a) at the time of master-initiation of DNA replication, points separated by 1.1-2.2x10(7) daltons of single strand DNA may initiate DNA synthesis. b) among these, only selected groups of replicons actually proceed in DNA replication at this time, while others appear to hold (later temporal sets of replicons). The origins of the ones that proceed in replication are separated from each other by a distance corresponding to 1.1-2.x10(7) daltons. c) regions in actual replication are separated from each other by increasing distances (up to 1.5x10(8) daltons single strand DNA) at later times in S.     

DNA synthesis in a sub-nuclear preparation isolated from Physarum polycephalum

A sub-nuclear preparation capable of substantial levels of DNA synthesis $\text{in}\text{vitro}$ has been obtained from isolated S-phase nuclei of $\text{Physarum}\text{polycephalum}$. Nuclei were disrupted by gentle resuspension in a dextran-free medium followed by immediate addition of dextran to stabilize the liberated replication complex. Synthesis continues for at least 120 min, and appears to occur by a semi-discontinuous mechanism. Little DNA synthesis occurs in preparations obtained from G₂-phase nuclei.     

DNA replication in Physarum polycephalum: electron microscopic analysis of patterns of DNA replication in the presence of cycloheximide

DNA from synchronously replicating nuclei of Physarum polycephalum was studied electron microscopically after 15, 30, 60, and 90 or 120 min of replication in the presence or absence of the protein synthesis inhibitor cycloheximide. The replication-loop size-distribution showed that replication fork progression is severely retarded in the presence of cycloheximide. Analysis of replication-loop frequency showed a similar pattern in control and cyclo-heximide-treated samples, with an increase from 15 to 30 and 60 min. This suggests, surprisingly, that initiations of new replicons either may not be inhibited by cycloheximide or, alternatively, that all initiations have already taken place at the very start of S-phase. The latter conclusion is favored in the light of previous results in our laboratory, discussed here.     

Endonuclease activity in nuclei of Physarum polycephalum. Partial purification and characterization.

An endonuclease, present in the microplasmodia of Physarum polycephalum, has been partially purified from isolated nuclei by DEAE-cellulose and Sephadex G-75 chromatography. 1. The endonuclease produced single-strand scissions in double-stranded DNA which resulted in the generation of 5'-phosphoryl and 3'-hydroxyl termini. No activity was observed with single-stranded DNA as substrate. 2. The pH optimum was approximately 8.5. 3. Divalent cations were essential for enzyme activity. MnCl2 and MgCl2 gave maximal activity. CaCl2, ZnCl2 or CoCl2 did not activate the enzyme. 4. The endonuclease activity was highly sensitive to monovalent cations. 5. Endonuclease activity was found in two forms after gel filtration: an activity in a homogeneous peak with a molecular weight of approx. 20 000, and an activity that had a heterogeneous molecular weight and which was isolated in a complex with DNA. A possible function of the endonuclease in DNA replication is discussed.