Delta-elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA.

[5'-32P]pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and [d(-)-1',2'-3H, 5'-32P]DNA, containing AP sites labelled with 3H in the 1' and 2' positions of the base-free deoxyribose [d(-)] and with 32P 5' to this deoxyribose, were used to investigate the yields of the beta-elimination and delta-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3'-phosphatase activity of T4 polynucleotide kinase, of the 3'-phosphate resulting from the beta delta-elimination. Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-elimination, hydrolysis of 3'-phosphate, DNA polymerization and ligation. Spermine, in one experiment, and Escherichia coli formamidopyrimidine: DNA glycosylase, in another experiment, were used to catalyse the first and second steps (beta-elimination and delta-elimination). These repair pathways, involving a delta-elimination step, may be operational not only in E. coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.     

Drosophila apurinic/apyrimidinic DNA endonucleases. Characterization of mechanism of action and demonstration of a novel type of enzyme activity

Two species of apurinic/apyrimidinic (AP) endonuclease have been purified approximately 400-fold from extracts of Drosophila embryos. AP endonuclease I, which flows through phosphocellulose columns, has an apparent subunit molecular weight of 66,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas AP endonuclease II, which is retained by phosphocellulose, has a subunit molecular weight of 63,000. The molecular weight determinations were made possible in part by the finding that both Drosophila enzymes, along with Escherichia coli endonuclease IV, cross-react with an antibody prepared toward a human AP endonuclease (Kane, C. M., and Linn, S. (1981) J. Biol. Chem. 256, 3405-3414). The nature of phosphodiester bond breaks produced by the two partially purified AP endonucleases from Drosophila have been investigated. Nicks introduced into partially depurinated PM2 DNA by Drosophila AP endonuclease I did not support DNA synthesis by E. coli DNA polymerase I, whereas nicks created by AP endonuclease II were able to support DNA synthesis, but at a rate far less than that observed for nicks introduced by E. coli endonuclease IV. The priming activity of DNA incised by either of the Drosophila enzymes can be enhanced, however, by an additional incubation with E. coli endonuclease IV, which is known to cleave depurinated DNA on the 5'-side of an apurinic site. These results suggest that the Drosophila enzymes cleave depurinated DNA on the 3'-side of the apurinic site. This suggestion was strengthened by the observation that the combined action of AP endonuclease II and E. coli endonuclease IV resulted in the removal of [32P]dAMP from partially depyrimidinated [dAMP-5'-32P,uracil-3H]poly(dA-dT). Taken together, these results propose that Drosophila AP endonuclease II produces 3'-deoxyribose and 5'-phosphomonoester nucleotide termini. Conversely, the absolute inability to detect priming activity for DNA cleaved by AP endonuclease I alone suggested a different mechanism, possibly the formation of a deoxyribose-3'-phosphate terminus. When apurinic DNA cleaved by AP endonuclease I was subsequently treated with bacterial alkaline phosphatase, DNA synthesis was now detected at levels similar to that observed for AP endonuclease II alone. Additionally, DNA nicked by AP endonuclease I was susceptible to 5'-end labeling by polynucleotide T4 kinase without prior phosphomonoesterase treatment. These results suggest that AP endonuclease I forms deoxyribose 3'-phosphate and 5'-OH termini upon cleaving depurinated DNA.     

Stimulation of phospholipase A2 activity by high osmotic pressure on cholesterol-containing phospholipid vesicles

Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 10(7) M-1 at 4 degrees C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Production of dissolved macromolecules by ambient autotrophic and heterotrophic microbial populations was measured in a eutrophic Florida reservoir by in situ labeling with various radioactive substrates. When [3H]thymidine was used as the precursor, production of labeled dissolved DNA, RNA, and protein was observed. The rate of production of labeled dissolved macromolecules was 3.1% the rate of cellular incorporation of [3H]thymidine, and the production of dissolved DNA represented 2.3% the rate of cellular DNA incorporation. Microautotrophic populations labeled with NaH[14C]CO3 produced dissolved RNA and protein at rates of 0.24 and 0.11 micrograms of C/liter per h, respectively, or 1.8% the total rate of carbon fixation, with no measurable dissolved DNA production. In an attempt to specifically label phytoplankton DNA, samples were incubated with [3H]adenine or 32Pi in the presence and absence of the photosynthetic inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Although DCMU inhibited 14C fixation by approximately 99%, this antimetabolite had only a slight effect on [3H]adenine incorporation and no effect on 32P incorporation into cellular macromolecules. Significant amounts of dissolved DNA were produced in both [3H]adenine and 32Pi incubations, but again DCMU had no effect on the production rates. These results indicate that actively growing populations of both phytoplankton and bacterioplankton produced dissolved RNA and protein, while only active bacterioplankton produced measurable quantities of dissolved DNA. Dead or senescent phytoplankton may have produced dissolved DNA, but would not be measured in the relatively short incubations used. These findings also indicate that [3H]adenine and 32Pi primarily labeled heterotrophic bacterioplankton and not phytoplankton in this environment.     

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.     

Effect of N-trifluoroacetyladriamycin-14-valerate on [3H]thymidine uptake and DNA synthesis of human lymphoma cells

The effects of N-trifluoroacetyladriamycin-14-valerate on the uptake of [3H]thymidine and its incorporation into DNA of human P3HR-1 lymphoma cells were studied. In the absence of the drug, at 0 degrees C, [3H]thymidine was transported into the cells but not incorporated into DNA, as determined by both the trichloroacetic acid-soluble and -precipitable counts obtained with the cells. At 37 degrees C, [3H]thymidine was readily transported into the cells and incorporated into DNA. In the presence of the drug, both [3H]thymidine uptake (as shown by acid-soluble counts) and the amount of its incorporation into acid-precipitable materials were markedly reduced. However, the uptake of [3H]thymidine at 0 degrees C was found to be equally sensitive to drug inhibition as at 37 degrees C. The incorporation at 37 degrees C of [3H]thymidine into acid-precipitable materials of the cells, which had been prelabeled at 0 degrees C with [3H]thymidine, was found to be insensitive to inhibition by the drug. The in vitro activities of DNA polymerases alpha and beta purified from human P3HR-1 cells were also found not to be susceptible to inhibition. Nuclei purified from cells pretreated with the drug continued to synthesize DNA. The cytofluorograms of the cells treated with the drug indicated that the treated cells accumulated at the G2/M phase, whereas the S phase of the cells was not arrested. These results suggest that N-trifluoroacetyladriamycin-14-valerate inhibits [3H]thymidine uptake but not cellular DNA synthesis in human P3HR-1 lymphoma cells.     

Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro.

In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.     

Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

Addition of platelet-activating factor (PAF) to cells doubly labeled with [14C]glycerol plus [3H]arachidonic acid resulted in a transient decrease of [14C]glycerol-labeled phosphatidylinositol (PI) and a transient increase of [14C]glycerol-labeled lysophosphatidylinositol (LPI). [3H]Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The 3H/14C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of [14C]glycerol-labeled DG paralleled the loss of triacyl [14C]glycerol and the 3H/14C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- [3H]inositol-prelabeled cells, PAF induced a transient decrease of [3H]phosphatidylinositol-4,5-bis-phosphate (TPI) and [3H]phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with 32Pi induced a transient decrease of [32P]polyphosphoinositides at 20 sec to 1 min. [32P]LPI appeared within 10 sec after stimulation and paralleled the loss of [32P]PI. [3H]Inositol triphosphate, [3H]inositol diphosphate, and [3H]inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.     

Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.     

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.     

Sequential degradation of choline phosphoglycerides by phospholipase D and phosphatidate phosphohydrolase in dibutyryl cAMP-differentiated U937 cells.

Dibutyryl-cAMP-differentiated U937 cells incorporate alkyllyso-sn-glycero-3-[32P]phosphocholine (alkyllyso-[32P]GPC) into cellular alkylacyl-sn-glycero-3-phosphocholine (alkylacyl-GPC). Upon stimulation with fMet-Leu-Phe (fMLP), recombinant C5a, or phorbol 12-myristate 13-acetate (PMA), these cells produce alkylacyl-sn-glycero-3-[32P]phosphate (alkylacyl-[32P]GP). In the presence of ethanol (0.5%), alkylacyl-sn-glycero-3-[32P]phosphoethanol (alkylacyl-[32P]GPet) is also formed with a concomitant reduction in alkylacyl-[32P]GP accumulation. Because cellular ATP is not labeled with 32P, alkylacyl-[32P]GP and alkylacyl-[32P]GPet must be formed by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Activation by receptor agonists, but not by PMA, requires extracellular Ca2+ and is augmented by cytochalasin B pretreatment. Upon stimulation, dibutryl cAMP-differentiated U937 cells labeled with alkylacyl-[32P]GPC produce [32P]PO4 but not [32P]phosphocholine. Furthermore, when these cells were labeled in alkylacyl-GPC by incubation with [3H]alkyllyso-GPC and then stimulated, [3H]alkylacyl-glycerol ([3H]alkylacyl-Gro) is produced with a time-course similar to that of [32P]PO4 formation and coincident with the decline in alkylacyl-GP accumulation. These results demonstrate that alkylacyl-GP formed by PLD is dephosphorylated by phosphatidate phosphohydrolase to produce PO4 and alkylacyl-Gro. Upon stimulation with fMLP or C5a, U937 cells labeled in diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) by incubation with [3H]acyllyso-GPC generate [3H]diacyl-GP, [3H]diacyl-GPEt, and [3H]diacyl-Gro with kinetics similar to those for the generation of the [3H]alkyl products. Thus, in differentiated U937 cells stimulated with receptor agonists, both alkylacyl-GPC and diacyl-GPC are sequentially metabolized by PLD and phosphatidate phosphohydrolase.     

Reutilization of phosphatidylglycerol and phosphatidylethanolamine by the pulmonary surfactant system in 3-day-old rabbits

Developing rabbits reutilize the phosphatidylcholine of surfactant with an efficiency of about 95%. The efficiency of reutilization of other components of surfactant have not been determined. 3-day-old rabbits were injected intratracheally with [3H]dipalmitoylphosphatidylcholine (DPPC) mixed with unlabeled natural surfactant and either disaturated [32P]phosphatidylglycerol (DSPG) or [14C]dipalmitoylphosphatidyl-ethanolamine (DPPE). The recovery of [3H]DPPC, [14C]DPPE, and [32P]DSPG in the alveolar wash was measured at different times after injection. By plotting the ratio of [32P]DSPG to [3H]DPPC or [14C]DPPE to [3H]DPPC counts/min in the alveolar wash vs. time after injection we showed that these two phospholipids are reutilized less efficiently than phosphatidylcholine. Based on other studies, several assumptions were made about the kinetics of surfactant phosphatidylethanolamine and phosphatidylglycerol. From the slopes of the semilog plots of total [14C]DPPE and total [32P]DSPG counts/min in the alveolar wash vs. time and these assumptions, we determined that these two phospholipids were reutilized at an efficiency of only 79%.     

Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [3H]Thd, [14C]orotate or with inorganic 32Pi. Expression of incorporated radioactivity as log10% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [3H]Thd was asymmetrically distributed among the isostichs, i.e., 3H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., T/C ratios for the first 10 isostichs averaged 1.43 +/- 0.08 and 1.28 +/- 0.05, depending on the method of analysis; furthermore, the T/C ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [3H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [3H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecular weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [14C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [3H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of 14C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from 32Pi, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since 32P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of 32P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the 'replitase' concept for channeling dNTPs to replicating forks.     

Mechanism of action of Moloney murine leukemia virus RNA-directed DNA polymerase associated RNase H (RNase H I)

The mechanism of action of the ribonuclease H (RNase H) activity associated with Moloney murine leukemia virus RNA-directed DNA polymerase (RNase H I) and the two-subunit (alpha beta) form of avian myeloblastosis virus DNA polymerase were compared by utilizing the model substrate (A)n.(dT)n and polyacrylamide gel electrophoresis in 7 M urea to analyze digestion products. Examination on 25% polyacrylamide gels revealed that a larger proportion of the RNase H I oligonucleotide products generated by limited digestion of [3H](A)(1100).(dT)n were acid insoluble (15-26 nucleotides long) than acid soluble (less than 15 nucleotides long), while the opposite was true for products generated by alpha beta RNase H. RNase H I was capable of attacking RNA in RNA.DNA in the 5' to 3' and 3' to 5' directions, as demonstrated by the use of [3H,3'- or 5'-32P](A)(380).(dT)n and cellulose--[3H](A)n.(dT)n. Both RNase H I and alpha beta RNase H degraded [3H]-(A)n.(dT)n with a partially processive mechanism, based upon classical substrate competition experiments and analyses of the kinetics of degradation of [3H,3'- or 5'-32P](A)(380).(dT)n. That is, both enzymes remain bound to a RNA.DNA substrate through a finite number of hydrolytic events but dissociate before the RNA is completely degraded. Both RNase H I and alpha beta RNase H were capable of degrading [14C](A)n in [3H](C)n-[14C](A)n-[32P](dA)n.(dT)n, suggesting that retroviral RNase H is capable of removing the tRNA primer at the 5' terminus of minus strand DNA at the appropriate time during retroviral DNA synthesis in vitro.     

Gaps in DNA synthesized by ultraviolet light-irradiated WI38 human cells.

DNA replication in ultraviolet-irradiated human cells was examined by treatment of the extracted DNA with a single-strand specific endonuclease from Neurospora crassa. WI38 cells were uniformly labeled with 32Pi for two generations before irradiation and then labeled with [3H]thymidine after irradiation. The isolated DNA was sedimented in neutral sucrose gradients after incubation with the endonuclease. The endonuclease treatment had no effect on the sedimentation profiles of either [32P]DNA or [3H]DNA from unirradiated control cultures. The endonuclease treatment also did not significantly alter the profile of [32P]DNA from irradiated cultures but did introduce breaks in the 3H pulse-labeled DNA synthesized after irradiation. These results indicate that DNA synthesis after ultraviolet irradiation proceeds in such fashion that gaps are formed along the newly made strand, leaving regions of single strandness in template DNA. As replication proceeds these gaps disappear and 2 h after irradiation (100-250 ergs/mm2) they are barely detectable by the endonuclease assay.     

Nucleosomes from normal and regenerating rat liver

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.     

Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S2 receptor sites

Upon stimulation with serotonin of washed human platelets prelabeled with [32P]orthophosphate, we found an approximately 250% increase in [32P]phosphatidic acid (PA) formation, a small decrease in [32P]phosphatidylinositol 4,5-bisphosphate, and a concomitant increase in [32P]phosphatidylinositol 4-phosphate. Using [3H]arachidonate for prelabeling, [3H]diacylglycerol accumulated transiently at 10 s after addition of the agonist, [3H]PA increased but to a lower extent compared to 32P-labeled lipid, and the formation of both [3H]polyphosphoinositides increased. The serotonin-induced dose-dependent changes in [32P]PA correlate with its effect on the changes in slope of aggregation of platelets. The potency of 13 drugs to antagonize the serotonin-induced PA formation closely corresponds to both their potency to inhibit platelet aggregation and their binding affinity for serotonin-S2 receptor sites. It is suggested that at least part of the signal transducing system following activation of the serotonin-S2 receptors involves phospholipase C catalyzed inositol lipid breakdown yielding diacylglycerol which is subsequently phosphorylated to PA.     

Phosphatidylcholine hydrolysis by phospholipase D determines phosphatidate and diglyceride levels in chemotactic peptide-stimulated human neutrophils. Involvement of phosphatidate phosphohydrolase in signal transduction

Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine (alkyl-PC) with 32P by incubation with alkyl-[32P]lysoPC. Upon stimulation with the chemotactic peptide, formylMet-Leu-Phe (fMLP), these 32P-labeled cells produce 1-O-alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) and, in the presence of ethanol, 1-O-alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Because the cellular ATP contains no 32P, alkyl-[32P]PA and alkyl-[32P]PEt must be formed from alkyl-[32P]PC by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Analyses of the sn-1 bonds by selective hydrolysis and mass measurements reveal that the PA and PEt formed during stimulation contain both ester and ether bonds with distributions similar to that in the endogenous PC. Furthermore, in neutrophils labeled in alkyl-[32P]PC, the specific activities of the diradyl-PA and diradyl-PEt formed during stimulation are similar to that of diradyl-PC. These results demonstrate that the fMLP-induced PLD utilizes diradyl-PC as the major substrate. It is further concluded that, at early times (30 s), PA and PEt are both formed almost exclusively by PLD. Following stimulation with fMLP, neutrophils double-labeled in alkyl-PC by incubation with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC generate [3H]alkyl-DG and [32P]orthophosphate [( 32P]PO4) with superimposable kinetics, indicating degradation of PA by a phosphohydrolase. Generation of [3H]alkyl-DG and [32P]PO4 lags behind PA formation and parallels the decline in PA accumulation. In addition, generation of both [3H]alkyl-PA and [3H]alkyl-DG requires extracellular Ca2+ and cytochalasin B. Furthermore, the phosphohydrolase inhibitor, propranolol, decreases both [3H]alkyl-DG and [32P]PO4 while increasing [3H]alkyl-PA and not altering [3H]alkyl-PEt. Moreover, the decreases in DG are accounted for by increases in PA. These results demonstrate that PLD-derived alkyl-PA is degraded by a phosphohydrolase to produce alkyl-DG. DG formed during stimulation contains both ester and ether-linked species and this DG formation is inhibited completely by propranolol. Upon stimulation, alkyl-[32P]PC-labeled neutrophils do not produce [32P]phosphocholine, suggesting that PC is not hydrolyzed by phospholipase C. In addition, PA is formed in amounts sufficient to account for all of the DG formed during stimulation. It is concluded that the DG formed during fMLP stimulation is derived almost exclusively from PC via the PLD/PA phosphohydrolase pathway.