Conversion of dNTP to dNMP dependent on DNA synthesis in isolated Yoshida sarcoma nuclei

Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one.     

Low-angle X-ray scattering analysis of the Thermoplasma acidophilum nucleoprotein subunit

A DNA-protein complex isolated from Thermoplasma acidophilum has been examined using low-angle X-ray scattering measurements. In agreement with the results of electron-microscopic studies a diamter of 5.5 nm is deduced. Finally, a simplified model of the DNA-protein particles is discussed postulating a kinked DNA.     

DNA fork displacement rates in Bloom's syndrome fibroblasts

DNA fork displacement rates were measured in three lines of Bloom's syndrome cells and in a normal diploid fibroblast line. Fork displacement rates in Bloom's cells were approx. 55–65% of the rate in normal fibroblasts.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Gamma-ray induced inhibition of DNA synthesis in ataxia telangiectasia fibroblasts is a function of excision repair capacity

The extent of the deficiency in γ-ray induced DNA repair synthesis in an ataxia telangiectasia (AT) human fibroblast strain was found to show no oxygen enhancement, consistent with a defect in the repair of base damage. Repair deficiency, but not repair proficiency, in AT cells were accompanied by a lack of inhibition of DNA synthesis (replicon initiation) neither γ-rays or the radiomimetic drug bleomycin. Experiments with 4-nitroquinoline 1-oxide indicated that lack of inhibition was specific for radiogenic type damage. Thus excision repair, perhaps by DNA strand incision or chromatin modification, appears to halt replicon initiation in irradiated repair proficient cells whereas in repair defective AT strains this putatively important biological function is inoperative.     

Unusual base sequence arrangement in phage phi 29 DNA.

Susceptibility of Bacillus subtilis phage phi 29 DNA to 34 different restriction endoculceases was determined. Three enzymes, BglI, XbaI and BstEII, were found to cleave phi 29 DNA only once at specific sites. The sites of these single cleavages have been mapped. Thirteen enzymes did not cut phi 29 DNA. phi 29 HindIII DNA fragments inserted into pBR313 plasmid and propagated in Escherichia coli, were resistant to these restriction endonucleases. This result suggests that the insusceptibility is due to the absence of the nucleotide sequences on phi 29 recognized by the enzymes, and not to the presence of modified nucleotides.     

Cytological changes and DNA and protein synthesis in parthenogenetically activated sea urchin eggs

Summary The present study deals with cytological observations, DNA and protein synthesis in artificially activated sea urchin eggs. The eggs were activated by means of Loeb's double treatment with butyric acid and hypertonic sea water. Most of the eggs ofHemicentrotus pulcherrimus divided when the chromosomes duplicated after formation of the first monaster and other eggs divided at a later cell cycle. In the eggs ofTemnopleurus toreumaticus, however, haploid division at the first cell cycle was observed predominantly.Activated eggs that were treated for 25 min with hypertonic sea water showed a marked uptake of³H-thymidine during the two periods of 30–40 min and 90–100 min after the double treatment. These periodic changes in the³H-thymidine uptake paralleled morphological changes within the nucleus. However, these periods of increased uptake were not observed in the eggs treated with hypertonic sea water for 60 min. During exposure to hypertonic sea water, the³H-thymidine-uptake by eggs activated with butyric acid decreased gradually. When the uptake of¹⁴C-valine by eggs was measured, a very low level was seen in unfertilized eggs. The level of uptake increased strikingly when the eggs were activated with butyric acid but was suppressed by the hypertonic treatment. However, removal of the eggs to sea water allowed the uptake to return to the former high level. This pattern suggests that the hypertonic treatment has an inhibitory effect on the synthesis of protein (or enzymes) which obstruct cleavage induction.     

Effects of aphidicolin on retrovirus DNA synthesis in vivo

Renaturation of $\text{Aequorea}$ green-fluorescent protein (A-GFP) was achieved for the first time following denaturation in guanidine-HCl or acid. Denaturation was accompanied by the concerted loss of visible fluorescence, alteration of absorption characteristics, and large negative deflection of CD signal in the far UV. Dialysis of a guanidine-denatured sample at pH 8 resulted in 64% renaturation (return to native absorption) and neutralization of an acid-denatured sample restored 90% of the native absorption. Renatured GFP is highly fluorescent and indistinguishable from native GFP with respect to the shape of excitation and emission spectra. Both native and denatured proteins exhibit resistance to trypsin hydrolysis and have identically broad pH and heat stability profiles, all of which suggest full renaturation.