Role In DNA Synthesis Research Articles

Correlation between levels of ferritin and the iron-containing component of ribonucleotide reductase in hydroxyurea-sensitive, -resistant, and -revertant cell lines

The reduction of ribonucleotides to deoxyribonucleotides, a rate-limiting step in DNA synthesis, is catalyzed by ribonucleotide reductase. This enzyme is composed of two components, M1 and M2. Recent work has shown that inhibition of ribonucleotide reductase by the antitumor drug hydroxyurea leads to a destabilized iron centre in protein M2. We have examined the relationship between the levels of ferritin, the iron storage protein, and the iron-containing M2 component of ribonucleotide reductase. These studies were carried out with hydroxyurea-sensitive, -resistant, and -revertant cell lines. Hydroxyurea-resistant mouse L cells contained M2 gene amplification and elevated levels of enzyme activity, M2 message, and total cellular M2 protein concentration. Hydroxyurea-revertant cells exhibited a wild-type M2 gene copy number, and approximately wild-type levels of enzyme activity, M2 message, and M2 protein concentration. In addition, we observed that the hydroxyurea-resistant cells possessed elevated levels of L-chain ferritin message and total cellular H-chain ferritin protein when compared to wild-type cells. In contrast, the revertant cell population contained approximately wild-type levels of ferritin mRNA and protein. In keeping with these observations, obtained with mouse L cells, was the finding that hydroxyurea-resistant Chinese hamster ovary cells with increased ribonucleotide reductase activity exhibited elevated expression of both ferritin and M2 genes, which declined in drug-sensitive revertant hamster cell lines with decreased levels of ribonucleotide reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of DNA synthesis in the responses of fetal rat calvariae to cortisol

To determine the extent to which the effects of cortisol on collagen synthesis in 21 day fetal rat calvariae are linked to its effects on cell replication, calvariae were cultured for 24-72 h with 0.1 and 1 microM cortisol in the presence or absence of 1 mM hydroxyurea (HU) or 30 microM aphidicolin (APC), inhibitors of DNA synthesis. The incorporation of [3H]proline into collagenase-digestible protein (CDP) and [3H]thymidine into DNA were measured during the last 2 h of culture. At 24 h HU and APC decreased thymidine incorporation by greater than 90%, and this remained low for the duration of culture. In contrast, cortisol reduced thymidine incorporation by only 44% at 72 h. Although cortisol caused a 24 h stimulatory effect and a 48 and 72 h inhibitory effect on CDP labeling and the percentage of collagen being synthesized (PCS), HU, and APC had no effect on basal CDP labeling or PCS over the 72 h culture period. Cortisol caused parallel alterations in the steady-state levels of alpha-1(I) procollagen mRNA, suggesting that its effects occur at the pretranslational level. At 24 h HU and APC did not prevent the stimulatory effect of cortisol on CDP labeling and PCS. At 48 h the inhibitory effects of cortisol on CDP labeling and PCS were observed in the presence of APC but not in the presence of HU. At 72 h the inhibitory effects of cortisol on CDP labeling and PCS were still observed in the presence of HU and APC.(ABSTRACT TRUNCATED AT 250 WORDS)

Targeting iron-dependent DNA synthesis with gallium and transferrin-gallium.

While iron is essential for numerous intracellular processes, its critical role in DNA synthesis relates to the activity of the iron-containing M2 subunit of ribonucleotide reductase, the enzyme responsible for the synthesis of deoxyribonucleotides. Gallium, a metal which resembles iron with respect to transferrin (Tf) binding, cellular uptake by the Tf receptor and incorporation into ferritin, blocks the cellular uptake of iron and inhibits cell growth. Exposure of HL60 cells to Tf-gallium (Ga) results in decreased deoxyribonucleotide synthesis and a diminution in the electron spin resonance (ESR) spectroscopy signal of ribonucleotide reductase, findings consistent with inhibition of this enzyme. In the present study, Ga nitrate blocked the uptake of 59Fe by L1210 cells and inhibited their proliferation. The ribonucleotide reductase M2 subunit ESR signal in cell cytoplasmic extracts was markedly inhibited in Ga-treated cells; however, the signal was restored to normal within 10 min of exposure of these cytoplasmic extracts to ferrous ammonium sulfate. These results confirm that Ga inhibits DNA synthesis by specifically limiting the amount of intracellular iron needed for the activity of the M2 subunit of ribonucleotide reductase. Further studies utilizing HL60 cells made resistant to Ga showed that these cells were also more resistant to growth inhibition by an anti-Tf receptor monoclonal antibody and deferoxamine. Ga blocks cell growth through inhibition of iron-dependent DNA synthesis. Cells appear to overcome the effects of Ga through compensatory mechanisms involving cellular iron metabolism.

Analysis of the binding sites for the varicella-zoster virus gene 51 product within the viral origin of DNA replication

The C-terminal 322 amino acids of the varicella-zoster virus (VZV) gene 51 product were expressed in Escherichia coli and shown to bind to specific DNA sequences within the VZV origin of DNA replication. The gene 51 product and its herpes simplex virus type 1 homolog (the UL9 protein) are capable of recognizing identical DNA sequences but the arrangement of binding sites within the origin regions of the two viruses differs. Three binding sites for the VZV gene 51 protein were identified within the VZV origin region and these lie in the same orientation and on the same side of the origin palindromic DNA sequence. DNA replication assays in transfected tissue culture cells demonstrated that the site closest to the palindrome is essential for origin activity whereas the most distal site is dispensable. The middle binding site may play an auxiliary role in DNA synthesis.

Amplification of the genes for both components of ribonucleotide reductase in hydroxyurea resistant mammalian cells

Ribonucleotide reductase catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, and is a rate-limiting step in the synthesis of DNA. The enzyme consists of two dissimilar subunits usually called M1 and M2. The antitumor agent, hydroxyurea, is a specific inhibitor of DNA synthesis and acts by destroying the tyrosyl free radical of the M2 subunit of ribonucleotide reductase. Two highly drug resistant cell lines designated H R-15 and H R-30 were isolated by exposing a population of mouse L cells to increasing concentrations of hydroxyurea. H R-15 and H R-30 cells contained elevated levels of ribonucleotide reductase activity, and were 68 and 103 times, respectively, more resistant than wild type to the cytotoxic effects of hydroxyurea. Northern and Southern blot analysis indicated that the two drug resistant lines contained elevated levels of M2 mRNA and M2 gene copy numbers. Similar studies with M1 specific cDNA demonstrated that H R-15 and H R-30 cell lines also contained increased M1 message levels, and showed M1 gene amplification. Mutant cell lines altered in expression and copy numbers for both the M1 and M2 genes are useful for obtaining information relevant to the regulation of ribonucleotide reductase, and its role in DNA synthesis and cell proliferation.

Does butyiphenyl-deoxyguanosine triphosphate differentially inhibit DNA polymerase α and δ activities in permeabilized HeLa cells?

In eukaryotic cells, two enzymes, DNA polymerases alpha and delta, are thought to play major roles in DNA synthesis. I have used butylphenyl dGTP (BuPdGTP), a potent inhibitor of purified DNA polymerase alpha, to assess the relative activities of these enzymes in two permeabilized cell systems. In both instances BuPdGTP eliminated all of the activity which was sensitive to aphidicolin. However, no conditions were found where BuPdGTP preferentially inhibited the synthesis of Okazaki fragments--the presumed products of DNA polymerase alpha activity. This implies that DNA polymerase activities on the two sides of the replication fork are unable to operate independently, being just two elements of the integrated replication machinery that undertakes DNA synthesis in permeabilized cells.

Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3' region of pol.

Twelve linker insertion mutations have been constructed in the 3' part of the pol gene of Moloney murine leukemia virus. This region of the Moloney murine leukemia virus genome encodes IN or p46pol, which is required for integration of the retroviral DNA into the host cell chromosome. Viral proteins synthesized by these mutants were used to pseudotype a neo-containing retroviral vector. Ten of twelve linker insertion mutant pseudotypes were unable to generate stable proviruses in infected mouse cells, as measured by the formation of G418-resistant colonies. Two mutants mapping at the 3' terminus of the IN-encoding region were competent for the formation of stable vector proviruses (hundreds of G418-resistant colonies per mutant pseudotype-infected plate). Representative linker insertion mutants were also tested for the ability to synthesize viral unintegrated DNA in newly infected cells. All assayed mutants were capable of synthesizing all normal forms of viral unintegrated DNA. The structure of integrated vector proviruses generated by defective and nondefective linker insertion mutants was also analyzed. All replication-competent mutants generated normal proviruses, while the few obtainable proviruses generated by replication-defective mutants were sometimes aberrant in structure. These results argue strongly (and confirm previous data) that the IN-encoding region of pol does not play a significant role in DNA synthesis, but is absolutely required for the formation of normal proviral DNA.

The Role of DNA Synthesis During Hypocotyl Elongation in Light and Dark

Fluorodeoxyuridine, an inhibitor of DNA synthesis, did not affect germination and post-germinative growth in the aerial part of lettuce and Haplopappus gracilis seedlings when grown in the light. In the dark, however, elongation of the hypocotyl was inhibited by fluorodeoxyuridine, strikingly in lettuce and only slightly in Haplopappus gracilis. This could imply that the controlling mechanism of hypocotyl elongation is in some cases related to DNA synthesis, either because mitotic processes (often little taken into account in considering hypocotyl growth) are involved in the elongation of hypocotyls only when they are grown in the dark, or because DNA synthesis affects cell elongation directly, or through the production of a greater number of endopolyploid cells in the dark. Using mainly autoradiographic and cytofluorimetric methods, these possibilities were tested. Besides lettuce (Lactuca sativa L. var. Great Lakes) and H. gracilis (Nutt.) Gray, radish (Raphanus sativus L. var. Tondo rosso quarantino) and soybean (Soya hyspida Sieb. and Zucc. var. Tokyo) seedlings were also studied. Fluorodeoxyuridine drastically inhibits cell elongation only when it is preceded or accompanied by mitotic or endomitotic events. Need for DNA synthesis during hypocotyl elongation, as well as during early post-germinative growth, seems to be of particular importance when endomitotic processes are involved.

Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites.

Pulse-chase experiments have revealed that cyclin, the auxiliary protein of DNA polymerase-delta, is stable during the transition from growth to quiescence in 3T3 cells. Immunoblotting together with immunofluorescence analysis has shown that the amount of cyclin after 24 h of quiescence is 30-40% of that of growing cells and that it presents a nucleoplasmic staining. Immunofluorescence studies show the existence of two populations of cyclin during the S phase, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent, and another that is associated to specific nuclear structures. By using antibromodeoxyuridine immunofluorescence to detect the sites of DNA synthesis, it was shown that the staining patterns of the replicon clusters and their order of appearance throughout the S phase are identical to those observed for cyclin. Two-dimensional gel analysis of Triton-extracted cells show that 20-30% of cyclin remains associated with the replicon clusters. This population of cyclin could not be released from the nucleus using high-salt extractions. This demonstrates that cyclin is tightly associated to the sites of DNA replication and that it must have a fundamental role in DNA synthesis in eukaryotic cells.

Participation of c-myc protein in DNA synthesis of human cells.

The protein product of oncogene c-myc is believed to be important in regulation of the cell cycle. However, its direct role in DNA synthesis has not been explored. Experiments presented here show that the addition of affinity-purified antibodies against the human c-myc protein to nuclei isolated from several types of human cells reversibly inhibited DNA synthesis and DNA polymerase activity of these nuclei. This suggests that c-myc encodes a protein that is functionally involved in DNA synthesis.

Increased aortic DNA synthesis precedes renal hypertension in rats. An obligatory step?

The rate of DNA synthesis was determined in rats with developing and established two-kidney, one clip renal hypertension. Rate of DNA synthesis was measured as [3H]thymidine incorporation into DNA per hour. After stenosis of the renal artery, blood pressure increased over a 2-week period. Five days after clipping, there was an increase in the rate of aortic DNA synthesis before an increase in blood pressure was detected, whereas there was no DNA effect in sham-operated animals. This difference in [3H]thymidine incorporation into aortic DNA could not be accounted for by alterations in thymidine pool sizes. The increase in DNA synthesis was still present 3 weeks after renal artery stenosis, although by that time blood pressure had plateaued. The role of DNA synthesis in the development of renal hypertension was investigated by determining whether inhibition of DNA synthesis with cytosine arabinoside could prevent the increase in blood pressure. Treatment of clipped rats with cytosine arabinoside for 5 days delayed the increase in blood pressure for more than 4 days, as compared with the effect of saline treatment in clipped rats. Although the possibility remains that some effect of cytosine arabinoside other than its effect on DNA synthesis could have influenced blood pressure, there were no differences in body weight, food intake, water intake, or urine output between cytosine arabinoside-treated and saline-treated rats with renal artery clips, and cytosine arabinoside treatment had no effect on blood pressure or body weight in normal rats. These results suggest that an increase in DNA synthesis may be an obligatory step in the genesis of renal hypertension.

DNA Polymerase-α: Enzymology, Function, Fidelity, and Mutagenesis

Publisher Summary This chapter focuses on the structure and catalytic properties of DNA polymerase-α, the major DNA polymerase in eukaryotic cells, because of its central role in DNA synthesis. a Until recently, progress in understanding the biochemistry of the enzyme was slow and the pathways to knowledge mimicked mainly studies in bacterial systems. Conditional mutants in DNA replication, so instrumental in isolating and defining the role of DNA replication proteins in bacteria, only recently became available from animal cells. In recent years, progress has been made in ascertaining the structure of this enzyme; mutant cell lines that contain an altered enzyme have been established and the gene coding for this enzyme has been assigned to a specific chromosome. Moreover, protocols to clone the gene for DNA polymerase-α are developing. The chapter assembles knowledge on the structure and catalytic properties of the polymerase and uses it as a framework for applying and evaluating the results of new molecular approaches to this problem.

Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits.

Consistent with its specialized role in DNA synthesis, the activity of ribonucleotide reductase is cell cycle-dependent, reaching its maximum during S-phase. This paper demonstrates, however, the levels of the two protein subunits, M1 and M2, of this enzyme vary independently of one another. The level of protein M1 was determined by use of a two-site monoclonal antibody-enzyme immunoassay and found to be constant throughout the cell cycle in bovine kidney MDBK cells. Pulse-chase experiments showed that the half-life of protein M1 was 15 h. This contrasts with our previous results demonstrating an S-phase-correlated increase in the concentration of protein M2 and a half-life of this subunit of 3 h. Therefore, ribonucleotide reductase is controlled during the cell cycle by the level of protein M2.

The role of DNA synthesis in peptidoglycan-induced generation of immunoglobulin-secreting cells in mice and humans

The requirement for DNA synthesis in peptidoglycan (PG)-induced activation of polyclonal antibodies has been studied. Inhibition of DNA synthesis with mitomycin C or hydroxyurea at the initiation of the cultures inhibited generation of over 90% of IgM- and IgG-secreting cells in PG-stimulated mouse splenocytes and of over 99.9% of IgM-, IgG- and IgA-secreting cells in PG-stimulated human peripheral blood lymphocytes. Inhibition of DNA synthesis 2 to 4 days after initiation of PG-stimulated cultures (in both mice and humans) caused an immediate decline in the numbers of Ig-secreting cells. These results demonstrate that the magnitude of PG-induced polyclonal antibody response depends on continued DNA synthesis and proliferation of Ig-secreting cells, and indicate that IgM-, IgG- and IgA-secreting cells in polyclonal activation may be actively cycling cells.

The genetic consequences of the Thy- mutation to CHO cells.

The behavior and regulation of dNTP pools in somatic cells has long led to speculation that they may play a role in DNA synthesis besides that of simple precursors [2, 20]. While controversy has developed concerning the relationship of dNTP pools to DNA synthesis [18, 19], observations from several laboratories provide convincing evidence that pool variations in somatic cells strongly influence spontaneous mutational rates and sensitivity to DNA damaging agents. The objective of the work in our laboratory has been to elucidate the basis of these effects on the accuracy of DNA synthesis by using defined somatic cell mutations which alter dNTP synthesis and pools. In this communication we summarize the properties of one of these mutant strains of Chinese hamster ovary (CHO) cells, called Thy-, and we speculate how the pool alterations produced by this metabolic defect might contribute to increased mutational rates and altered sensitivity to DNA damaging agents.

Hormonal stimulation of myoblast differentiation in the absence of DNA synthesis.

The role of DNA synthesis in the final stages of muscle cell differentiation has been a subject of controversy for more than a decade. In an attempt to resolve disagreements over the necessity for a unique (or "quantal") mitosis just prior to the conversion of proliferating myoblasts to form postmitotic myotubes, we have studied the effects of insulin and somatomedin on the stimulation of myoblast differentiation with or without DNA synthesis. Under conditions in which at least 95% of [3H]thymidine incorporation was blocked by cytosine arabinoside, there was a 5- to 10-fold increase in the extent of differentiation (determined as fusion or creatine kinase elevation) on addition of insulin or multiplication-stimulating activity. The effect of the hormones was on myoblast differentiation, not enzyme induction; insulin did not cause any increase in creatine kinase when it was added to performed myotubes. These studies were done using two different cell types, Yaffe's L6 cell line and Japanese quail myoblasts in serum-free media; we obtained similar results in both. Our results are not compatible with the view that a quantal mitosis is required at a late stage of muscle cell differentiation.

Abnormal deoxyuridine suppression test in congenital methylmalonic aciduria-homocystinuria without megaloblastic anemia: divergent biochemical and morphological bone marrow manifestations of disordered cobalamin metabolism in man

We studied two brothers (J.R. and M.R.) with the cobalamin D variant of congenital methylmalonic aciduria-homocystinuria, whose previously reported lack of megaloblastic anemia conflicted with current concepts of cobalamin's role in DNA synthesis and the "methyltetrahydrofolate (MTHF) trap" hypothesis. Both subjects were indeed hematologically normal, although J.R. had a mean corpuscular volume of 96 fl. However, both demonstrated abnormalities in the deoxyuridine suppression test. J.R. had an abnormal suppression value of 21.0% (normal less than 10%) that was correctable by adding hydroxocobalamin or folic acid in vitro but not MTHF. M.R. had normal suppression (8.9%), but demonstrated worsening (18.6%) when MTHF was added. J.R.'s classical deoxyuridine suppression pattern of cobalamin deficiency thus supports the trap hypothesis. However, his lack of comparable morphological changes suggests that impaired de novo thymidylate synthesis and the trap hypothesis, though valid, may not fully account for the megaloblastic maturation accompanying cobalamin deficiency. Equally noteworthy was the deleterious effect of MTHF on M.R.'s marrow, suggesting its potential usefulness as an in vitro "stress test" for latent cobalamin abnormality.

Abnormal deoxyuridine suppression test in congenital methylmalonic aciduria-homocystinuria without megaloblastic anemia: divergent biochemical and morphological bone marrow manifestations of disordered cobalamin metabolism in man

We studied two brothers (J.R. and M.R.) with the cobalamin D variant of congenital methylmalonic aciduria-homocystinuria, whose previously reported lack of megaloblastic anemia conflicted with current concepts of cobalamin's role in DNA synthesis and the “methyltetrahydrofolate (MTHF) trap” hypothesis. Both subjects were indeed hematologically normal, although J.R. had a mean corpuscular volume of 96 fl. However, both demonstrated abnormalities in the deoxyuridine suppression test. J.R. had an abnormal suppression value of 21.0% (normal less than 10%) that was correctable by adding hydroxocobalamin or folic acid in vitro but not MTHF. M.R. had normal suppression (8.9%), but demonstrated worsening (18.6%) when MTHF was added. J.R.'s classical deoxyuridine suppression pattern of cobalamin deficiency thus supports the trap hypothesis. However, his lack of comparable morphological changes suggests that impaired de novo thymidylate synthesis and the trap hypothesis, though valid, may not fully account for the megaloblastic maturation accompanying cobalamin deficiency. Equally noteworthy was the deleterious effect of MTHF on M.R.'s marrow, suggesting its potential usefulness as an in vitro “stress test” for latent cobalamin abnormality.

The role of putrescine in modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine.

The relationship between polyamine metabolism and DNA synthesis in regenerating liver was investigated using normal and 5-azacytidine-treated rats. The increase of DNA synthesis in regenerating rat liver was completely inhibited by administration of the analogue 1 h after partial hepatectomy. On the contrary, early and enhanced increase (modulation) of DNA synthesis was observed when the analogue was injected 24 h before partial hepatectomy. Changes in ornithine decarboxylase [EC 4.1.1.17] activity and the tissue levels of polyamines in liver remnants showed similar patterns in normal and 5-azacytidine-pretreated rats, except that the putrescine level in removed liver and the spermidine level in the S-phase were significantly higher in the drug-pretreated rats. On the other hand, a single injection of the analogue into intact rats evoked marked increase in hepatic ornithine decarboxylase activity followed by increase in the tissue level of putrescine, but not of spermidine or spermine. No similar increase in the enzyme activity was detected after injection of 5-aza-2'-deoxycytidine, which did not cause modulation of DNA synthesis. No modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine was observed when partial hepatectomy was carried out before increase of the hepatic level of putrescine. In addition, a positive and highly significant correlation was observed between the level of putrescine in the liver removed at partial hepatectomy and the extent of modulation of DNA synthesis in regenerating liver of rats pretreated with 5-azacytidine. These results suggest that putrescine plays an important role in modulation of DNA synthesis and support our previous proposal that putrescine may have at least two roles in DNA synthesis.

Induction of Mammary Tumors in Aging Rats by 7,12-Dimethylbenz[<italic>a</italic>]anthracene: Role of DNA Synthesis During Carcinogenesis<xref ref-type="fn" rid="FN2">2</xref>

Two routes of administration were used to test the susceptibility of the mammary gland of the rat to 7,12-dimethylbenz[a]anthracene (DMBA) carcinogenesis in relation to age of the tissue. In one series of experiments, 60-, 70-, 90-, 120-, 150-, and 200-day-old female noninbred Sprague-Dawley rats were given DMBA iv. In parallel experiments, rats of the same ages as those above were given DMBA by local application. Mammary tumors developed in 80-90% of the 60- and 70-day-old rats and in 40% of the 90-day-old rats. Rats 120 days old and older were completely "refractory" to DMBA. In contrast, all rats, irrespective of their ages, developed tumors when DMBA was applied locally. The DNA synthesis labeling index (LI) of mammary glands of rats in two series of experiments was examined by [3H]thymidine incorporation and autoradiography at 24, 48, and 72 hours after DMBA treatment. DMBA, given iv significantly inhibited DNA synthesis in mammary glands, but DMBA applied locally significantly increased the LI of the mammary glands. Thus the LI of the mammary glands in 60-day-old rats increased from 8% (control, untreated) to 16% (rats receiving local applications of DMBA), whereas the LI of the mammary glands in 150-day-old rats increased from 1.15% (control, untreated) to 8% (rats receiving local application of DMBA).