DNA Polymerase Alpha Activity Research Articles

Induction of Apoptosis in Metastatic Breast Cancer Cells: XV. Downregulation of DNA Polymerase-α - Helicase Complex (Replisomes) and Glyco-Genes.

In normal and cancer cells, successful cell division requires accurate duplication of chromosomal DNA. All cells require a multiprotein DNA duplication system (replisomes) for their existence. However, death of normal cells in our body occurs through the apoptotic process. During apoptotic process several crucial genes are downregulated with the upregulation of caspase pathways, leading to ultimate degradation of genomic DNA. In metastatic cancer cells (SKBR-3, MCF -7, and MDA-462), this process is inhibited to achieve immortality as well as overexpression of the enzymes for the synthesis of marker molecules. It is believed that the GSL of the lacto family such as LeX, SA-LeX, LeY, Lea, and Leb are markers on the human colon and breast cancer cells. Recently, we have characterized that a few apoptotic chemicals (cis-platin, L-PPMP, D-PDMP, GD3 ganglioside, GD1b ganglioside, betulinic acid, tamoxifen, and melphalan) in low doses kill metastatic breast cancer cells. The apoptosis-inducing agent (e.g., cis-platin) showed inhibition of DNA polymerase/helicase (part of the replisomes) and also modulated (positively) a few glycolipid-glycosyltransferase (GSL-GLTs) transcriptions in the early stages (within 2h after treatment) of apoptosis. These Lc-family GSLs are also present on the surfaces of human breast and colon carcinoma cells. It is advantageous to deliver these apoptotic chemicals through the metastatic cell surfaces containing high concentration of marker glycolipids (Lc-GSLs). Targeted application of apoptotic chemicals (in micro scale) to kill the cancer cells would be an ideal way to inhibit the metastatic growth of both breast and colon cancer cells. It was observed in three different breast cancer lines (SKBR-3, MDA-468, and MCF-7) that in 2h very little apoptotic process had started, but predominant biochemical changes (including inactivation of replisomes) started between 6 and 24h of the drug treatments. The contents of replisomes (replisomal complexes) during induction of apoptosis are not known. It is known that DNA helicase activities (major proteins catalyze the melting of dsDNA strands) change during apoptotic induction process. Previously DNA Helicase-III was characterized as a component of the replication complexes isolated from carcinoma cells and normal rapid growing embryonic chicken brain cells. Helicase activities were assayed by a novel method (combined immunoprecipitation-ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of nicked "ACT-DNA," by determining values obtained from +/- aphidicolin added to the incubation mixtures. Very little is known about the stability of the "replication complexes" (or replisomes) during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during replication, repair, and recombination processes. In all three breast carcinoma cell lines (SKBR-3, MCF-7, and MDA-468), a common trend, decrease of activities of DNA polymerase-alpha and Helicase-III (estimated and detected with a polyclonal antibody), was observed, after cis-platin- and L-PPMP-induced apoptosis. Previously our laboratory has documented downregulation (within 24-48h) of several GSL-GLTs with these apoptotic reagents in breast and colon cancer cells also. Perhaps induced apoptosis would improve the prognosis in metastatic breast and colon cancer patients.

Aberrant DNA Polymerase α Is Excluded from the Nucleus by Defective Import and Degradation in the Nucleus

DNA polymerase alpha is essential for the onset of eukaryotic DNA replication. Its correct folding and assembly within the nuclear replication pre-initiation complex is crucial for normal cell cycle progression and genome maintenance. Due to a single point mutation in the largest DNA polymerase alpha subunit, p180, the temperature-sensitive mouse cell line tsFT20 exhibits heat-labile DNA polymerase alpha activity and S phase arrest at restrictive temperature. In this study, we show that an aberrant form of endogenous p180 in tsFT20 cells (p180(tsFT20)) is strictly localized in the cytoplasm while its wild-type counterpart enters the nucleus. Time-lapse fluorescence microscopy with enhanced green fluorescent protein-tagged or photoactivatable green fluorescent protein-tagged p180(tsFT20) variants and inhibitor analysis revealed that the exclusion of aberrant p180(tsFT20) from the nucleus is due to two distinct mechanisms: first, the inability of newly synthesized (cytoplasmic) p180(tsFT20) to enter the nucleus and second, proteasome-dependent degradation of nuclear-localized protein. The nuclear import defect seems to result from an impaired association of aberrant de novo synthesized p180(tsFT20) with the second subunit of DNA polymerase alpha, p68. In accordance, we show that RNA interference of p68 results in a decrease of the overall p180 protein level and in a specific increase of cytoplasmic localized p180 in NIH3T3 cells. Taken together, our data suggest two mechanisms that prevent the nuclear expression of aberrant DNA polymerase alpha.

Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, -3, -8, and -9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ';Replication Complexes'' during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/- aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-alpha and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 alpha2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a alpha2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.

Effect of novel benzoylphenylurea derivatives on DNA polymerase alpha activity using the synthesome-based in vitro model system.

Six benzoylphenylurea (BPU) derivatives have been synthesized in Japan and extensively evaluated by the U.S. National Cancer Institute. They demonstrated potent antitumor activity in vitro against several cancer cell lines as well as in vivo against several tumor models. One of these agents, NSC639829, has now entered clinical trials. Studies have shown that these compounds are effective inhibitors of in vitro tubulin polymerization. The parent compound, NSC624548 (HO-221), has been shown to inhibit calf thymus DNA polymerase alpha activity. In this study we examined the effects of four BPU derivatives (NSC624548, NSC639828, NSC639829, and NSC654259) on the activity of the synthesome-associated DNA polymerase alpha, Escherichia coli DNA polymerase I, and calf thymus DNA polymerase alpha. Among the compounds tested, only NSC624548 and NSC639828 inhibited the activities of E. coli DNA polymerase I and calf thymus DNA polymerase alpha. Excess DNA polymerase I or DNA polymerase alpha dramatically reduced the inhibition produced by these compounds. NSC624548 and NSC639828 also showed inhibitory effects of the synthesome-associated DNA polymerase alpha similar to that produced upon using the purified E. coli and calf thymus enzymes. All of the four compounds did not show inhibitory effect on DNA polymerase delta. The similar pattern of inhibition these compounds exert on both the purified calf thymus and the synthesome-associated DNA polymerase alpha offers further support for the validity of the DNA synthesome as a novel in vitro model system for studying anticancer drug action.

Dissociation of DNA polymerase alpha-primase complex during meiosis in Coprinus cinereus.

Previously, the activity of DNA polymerase alpha was found in the meiotic prophase I including non-S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase alpha during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase alpha catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno-staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol alpha-primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol alpha-primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol alpha-primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase alpha-primase complex is altered during meiotic development.

Expression of two isoform mRNAs of nucleolar protein B23 during rat liver regeneration.

Nucleolar protein B23 exists in two isoforms designated as B23.1 and B23.2, differing only in their carboxy-terminal short sequences. To clarify the levels of protein B23 isoform mRNAs in the cell cycle, we have investigated the expressions of the two mRNAs during rat liver regeneration. Both of B23 isoforms transcripts increased after a partial hepatectomy. Peaks of the mRNAs were observed after 9 h (fivefold) with B23.1 and 12 h (twofold) with B23.2, respectively. These two peaks were slightly preceding the onset of DNA synthesis, which was revealed by the activity of DNA polymerase alpha. From these observations, important roles of both protein B23 isoforms in stimulation of DNA polymerase a activity during rat liver regeneration were suggested.

Human cell DNA replication is mediated by a discrete multiprotein complex.

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.

The role of DNA polymerase activity in human non-homologous end joining.

In mammalian cells, DNA double-strand breaks are repaired mainly by non-homologous end joining, which modifies and ligates two DNA ends without requiring extensive base pairing interactions for alignment. We investigated the role of DNA polymerases in DNA-PK-dependent end joining of restriction-digested plasmids in vitro and in vivo. Rejoining of DNA blunt ends as well as those with partially complementary 5' or 3' overhangs was stimulated by 20-53% in HeLa cell-free extracts when dNTPs were included, indicating that part of the end joining is dependent on DNA synthesis. This DNA synthesis-dependent end joining was sensitive to aphidicolin, an inhibitor of alpha-like DNA polymerases. Furthermore, antibodies that neutralize the activity of DNA polymerase alpha were found to strongly inhibit end joining in vitro, whereas neutralizing antibodies directed against DNA polymerases beta and epsilon did not. DNA sequence analysis of end joining products revealed two prominent modes of repair, one of which appeared to be dependent on DNA synthesis. Identical products of end joining were recovered from HeLa cells after transfection with one of the model substrates, suggesting that the same end joining mechanisms also operate in vivo. Fractionation of cell extracts to separate PCNA as well as depletion of cell extracts for PCNA resulted in a moderate but significant reduction in end joining activity, suggesting a potential role in a minor repair pathway.

Transcriptional Activation of Deoxyribonucleic Acid Polymerase α Gene Expression in MCF-7 Cells by 17β-Estradiol*

Treatment of MCF-7 human breast cancer cells with 17beta-estradiol (E(2)) results in increased DNA synthesis and cell proliferation and enhanced enzyme activities associated with purine/pyrimidine biosynthesis. The mechanism of enhanced DNA polymerase alpha activity was investigated by analysis of the promoter region of this gene. E(2) induced luciferase (reporter gene) activity in MCF-7 cells transfected with pDNAP1, pDNAP2, and pDNAP3 containing -1515 to +45, -248 to +45 and -116 to +45 inserts from the DNA polymerase alpha gene promoter, whereas no induction was observed with pDNAP4 (-65 to +45 insert). The induction response was dependent on cotransfection with estrogen receptor alpha (ER(alpha)), and transactivation was also observed with a mutant ER(alpha) that did not express the DNA-binding domain. Subsequent functional, DNA binding, and DNA footprinting studies showed that a GC-rich region at -106 to -100 was required for E(2)-mediated transactivation, and Sp1 protein, but not ER(alpha), bound this sequence. Transcriptional activation of DNA polymerase alpha by E(2) is associated with ER(alpha)/Sp1 action at a proximal GC-rich promoter sequence, and this gene is among a growing list of E(2)-responsive genes that are induced via ER(alpha)/Sp1 protein interactions that do not require direct binding of the hormone receptor to DNA.

Radiosensitivity of human squamous carcinoma cell lines is associated with amount of spontaneous DNA strand breaks.

We asked whether the constitutive level of DNA strand breaks (SBs) in four human squamous carcinoma cell lines is associated with their radiosensitivity, measured by the clonogenic assay. Because impairment in DNA replication and the action of endogenous deoxyribonucleases are two major sources of DNA strand breaks under normal cell metabolism, we also analyzed DNA polymerase and DNA ligase activities as well as the functional status of Poly(ADP-ribose) polymerase (PARP) and nucleolytic degradation of genomic DNA. We showed that the two relatively radioresistant cell lines, UM-SCC-1 and UT-SCC-5, had a statistically significant lower constitutive level of DNA SBs, as measured by DNA precipitation technique, compared with the two relatively radiosensitive cell lines, UM-SCC-14A and UT-SCC-9. We found that cell lines with a higher level of broken DNA tended to have a higher constitutive level of DNA polymerase alpha activity, measured by incorporation of [(3)H]dTTP in DNase I-activated DNA. UM-SCC-1, UT-SCC-5, and UM-SCC-14A did not show any difference in DNA ligase activity when a nicked oligonucleotide was used as substrate. The most radiosensitive cell line, UT-SCC-9, had a significantly lower ligation efficiency compared to the other three cell lines. The functional status of the PARP was the same in the four cell lines. Although none of the four cell lines showed a characteristic apoptotic or necrotic degradation of genomic DNA, when tested with the "plasmid rejoining assay," a significant degradation of the plasmid DNA in UT-SCC-9 was detected. We conclude that the high fraction of DNA SBs for UT-SCC-9, the most radiosensitive cell line, is most likely a consequence of low ligation efficiency combined with a relatively high DNA polymerase alpha activity and the nuclease degradation of DNA.

Galactosyldiacylglycerol, a mammalian DNA polymerase alpha-specific inhibitor from a sea alga, Petalonia bingbamiae.

The glycolipid galactosyldiacylglycerol (GDG), containing C16:0 and C18:1 fatty acids, was isolated from the sea alga Petalonia bingbamiae as a potent inhibitor of the activities of mammalian DNA polymerase alpha (pol. alpha). GDG, however, had no effect on pol. alpha from a fish or a higher plant. The inhibition of pol. alpha by GDG was dose-dependent with an IC50 value of 54 microM. The compound did not influence the activities of other replicative DNA polymerases such as mammalian pol. delta, or repair-related enzymes such as mammalian pol. beta. GDG also did not influence the activities of prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase, Taq DNA polymerase, DNA polymerases from the higher plant, cauliflower, or DNA metabolic enzymes such as calf thymus terminal deoxynucleotidyl transferase, human immunodeficiency virus type 1 reverse transcriptase and deoxyribonuclease 1. Kinetic analysis of the compound showed that pol. alpha was non-competitively inhibited with respect to both the DNA template and the nucleotide substrate. In this study, we demonstrated the structure-function relationship in the selective inhibition of pol. alpha by the glycolipid group.

The cytotoxicity of symmetrical and unsymmetrical bis(thiosemicarbazones) and their metal complexes in murine and human tumor cells.

A number of thiosemicarbazones have been tested previously and herein are included three bis(thiosemicarbazones) for comparison to the previous derivatives. In general the uncomplexed thiosemicarbazones were more potent in the cytotoxic screens than the bis(thiosemicarbazone) except in the murine L1210 and the human colon SW480 screens. Mode of action studies have only demonstrated slight differences in the effects of the two types of compounds on nucleic acid metabolism. The symmetrical and unsymmetrical bis(thiosemicarbazones) complexes of copper, nickel, zinc, and cadmium have been examined to compare them to the heterocyclic N(4)-substituted thiosemicarbazones metal complexes. These new derivatives demonstrated excellent activity against the growth of suspended lymphomas and leukemias although it should be pointed out that generally they were not as active as the copper complexes of N(4)-substituted thiosemicarbazones. Nevertheless, selected bis(thiosemicarbazones) complexes were active against the growth of human lung MB9812, KB nasopharynx, epidermoid A431, glioma UM-86, colon SW480, ovary 1-A9, breast MCK-7, and osteosarcoma Saos-2. In human HL-60 promyelocytic leukemia cells the complexes preferentially inhibited DNA and purine syntheses over 60 min. The regulatory enzyme of the de novo purine pathway, IMP dehydrogenase, appeared to be a major target of the complexes. However, minor inhibition of the activities of DNA polymerase alpha, PRPP-amido transferase, ribonucleotide reductase, and nucleoside kinases occurred over the same time period. No doubt these effects of the complexes on nucleic acid metabolism were additive since the d[NTP] pool levels were reduced after 60 min as was DNA synthesis. The symmetrical and unsymmetrical bis(thiosemicarbazones) and their metal complexes did not cause as severe DNA fragmentation as the heterocyclic N(4)-substituted thiosemicarbazone metal complexes; furthermore, their metabolic effects in the tumor cell were more focused on a single synthetic pathway.

DNA polymerases and Ki-67 nuclear antigen are induced in correlation with the resected mass of rat liver up to 90%.

We studied the regeneration potential by measuring induction of DNA polymerases in the remnant rat liver after a partial hepatectomy (PHx) that is maximal but compatible with survival. The regenerating rat liver was obtained after the 90% PHx. The induction of activities of DNA polymerase alpha, delta, and epsilon were measured after partial purification. The Ki-67 nuclear antigen was also detected histochemically. These parameters were compared with those after both 30% and 70% PHx. The 90% hepatectomy resulted in the strong inductions of DNA polymerase alpha, delta, and epsilon, at 48 h after operation, in association with increases in wet weight and total DNA in the remnant liver. The enzyme induction was much higher after 90% PHx than after 30% and 70% hepatectomy, in correlation with the resection volume. At 48 h after 90% hepatectomy, the Ki-67 positive cells increased up to 47.2% of hepatocytes in the remnant liver. The higher induction of replication enzymes by 90% hepatectomy reflects more cells entering mitogenic cell cycle, which supports the fast regeneration of the remnant liver. The number of proliferating hepatocytes is stringently controlled by an unknown mechanism sensing the mass of resected liver parenchyma.

Effects of gemcitabine and araC on in vitro DNA synthesis mediated by the human breast cell DNA synthesome.

Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis. We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects ofdFdCTP and araCTP on the synthesome-associated DNA polymerase alpha and delta activities. Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 microM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full-length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 microM dCTP, 3 microM dFdCTP and 60 microM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases alpha and delta. It was found that the drug concentrations required to inhibit 50% of the synthesome-associated DNA polymerase delta activity were much higher than those required to inhibit 50% of DNA polymerase alpha activity for both dFdCTP and araCTP. Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase alpha activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.

Damage to DNA and activity of nuclear DNA repair and replicative enzymes following N-nitrosodiethylamine treatment to rats.

Continuous administration in the drinking water of hepatocarcinogen N-nitrosodiethylamine (NDEA) to male rats (200 mg/L) for 60 days resulted in DNA damage in the form of single strand breaks. The damage, which is measured as a shift in the sedimentation of DNA in alkaline sucrose density gradients, was found to be maximum at the fourth week of treatment, and the sedimentation pattern of DNA was found to return to near normal size by the seventh week of NDEA treatment. Simultaneously, there were perturbations in the nuclear enzymes involved in DNA replication and repair. Activities of DNA polymerase beta, DNA ligase, and topoisomerase were found to increase in as early as the first week of NDEA treatment and reached the maximum at the fourth week, and thereafter declined to normal level by the eighth week of treatment. Concomitantly, the activities of DNA polymerase alpha, DNA primase, and RNA polymerase which were unaltered in the initial period of carcinogen treatment recorded a marked increase after sixth week of NDEA treatment. Results suggest that administration of NDEA inflicts DNA damage, which is manifested as increase in DNA repair enzymes in the initial period and activated DNA replicative enzymes at a later period, indicating the active proliferation of transformed cells.

Activity of DNA polymerase alpha in aging human fibroblasts.

During the past decade intense investigation has focused on cellular aging with the expectation of discovering factors that regulate the replication complex and contribute to the onset and progression of cellular aging. The most striking feature of cellular aging is the failure of sensing diploid cells to enter or complete S phase of the cell cycle. The G1/S phase transition is an initial critical step in the regulation of proliferation in eukaryotic cells, and significant advances have been made toward understanding the basic mechanisms of aging by identifying components of the macromolecular assemblies participating in the G1/S transition. These studies have identified multiple DNA polymerases and their accessory factors, and have provided important strategies for investigating the molecular events that contribute to aging processes. DNA replication, repair and recombination in eukaryotic cells require the action of a variety of DNA polymerases, at least six of which are known, alpha, beta, gamma, delta, epsilon, and zeta. Among them the highly conserved DNA polymerase alpha-primase (pol alpha-primase) is the only enzyme capable of initiating DNA replication at chromosomal origin sites and at sites of initiation of discontinuous synthesis of Okazaki fragments on the lagging side of the replication fork. Numerous protein factors that play strategic roles in DNA replication have been identified and the understanding of their regulation has been an important step for identifying the elements that are involved in, and possibly necessary for, governing cellular senescence and aging. In this review we summarize the current information regarding DNA pol alpha modulation during aging. We focus in particular on the coordinated actions of DNA pol alpha in the presence of other cellular proteins involved in the replication complex in the hope that understanding pol alpha interactions with components of the replication complex may provide insight into the mechanisms by which aging and age-related diseases occur.

Mode of action of thiocoraline, a natural marine compound with anti-tumour activity.

Thiocoraline, a new anticancer agent derived from the marine actinomycete Micromonospora marina, was found to induce profound perturbations of the cell cycle. On both LoVo and SW620 human colon cancer cell lines, thiocoraline caused an arrest in G1 phase of the cell cycle and a decrease in the rate of S phase progression towards G2/M phases, as assessed by using bromodeoxyuridine/DNA biparametric flow cytometric analysis. Thiocoraline does not inhibit DNA-topoisomerase II enzymes in vitro, nor does it induce DNA breakage in cells exposed to effective drug concentrations. The cell cycle effects observed after exposure to thiocoraline appear related to the inhibition of DNA replication. By using a primer extension assay it was found that thiocoraline inhibited DNA elongation by DNA polymerase α at concentrations that inhibited cell cycle progression and clonogenicity. These studies indicate that the new anticancer drug thiocoraline probably acts by inhibiting DNA polymerase α activity. © 1999 Cancer Research Campaign

Nucleolar protein B23.1 binds to retinoblastoma protein and synergistically stimulates DNA polymerase alpha activity.

Phosphorylated retinoblastoma protein and nucleolar protein B23 are putative stimulatory factors for DNA polymerase alpha. We showed that these two factors interacted with each other and stimulated the activity of DNA polymerase alpha synergistically. B23 exists in two isoforms designated as B23.1 and B23.2. While B23.1 bound to a retinoblastoma protein-conjugated column, B23.2 did not. These results indicate that B23.1 can directly bind to retinoblastoma protein. It was also shown that B23 was co-immunoprecipitated with both retinoblastoma protein and DNA polymerase alpha from a HeLa cell extract by monoclonal antibodies raised against these components. These results suggest that these three proteins exist as a complex in cells, at least in part. The simultaneous addition of both B23.1 and retinoblastoma protein caused stimulation of DNA polymerase alpha activity that is much higher than the sum of the stimulation by retinoblastoma protein and B23.1 alone. The maximal stimulation was attained at the molar ratio of DNA polymerase alpha/retinoblastoma protein/B23.1 = 1:1:12. Since B23 exists as a hexamer in solution, it may act as a stimulator of DNA polymerase alpha in a form of double-hexamer, in concert with the phosphorylated retinoblastoma protein.

Inhibition of eukaryotic dna polymerase alpha by persimmon (Diospyros kaki) extract and related polyphenols.

The effects of persimmon extract (Diospyros kaki) and related polyphenols on eukaryotic DNA polymerase alpha were examined. It was found that persimmon extract, epigallocatechin gallate, and epicatechin gallate strongly inhibited the activity of DNA polymerase alpha purified from calf thymus. Among these polyphenols, persimmon extract had the most potent effect on DNA polymerase alpha activity and the concentration of persimmon extract producing 50% inhibition of the activity was 0.191 microM. Persimmon extract showed a weaker effect on DNA polymerase beta and slightly inhibited primase and DNA polymerase I. The inhibition of DNA polymerase alpha by persimmon extract was competitive with the template-primer and noncompetitive with dTTP substrate. The Ki value of DNA polymerase alpha for persimmon extract was estimated to be 70 nM. Moreover, persimmon extract inhibited [3H]thymidine incorporation of human peripheral lymphocyte cells stimulated by PHA.

Action of a new mammalian DNA polymerase inhibitor, sulfoquinovosyldiacylglycerol.

We found and previously reported a new mammalian DNA polymerase inhibitor from a sea alga, Gigartina tenella, (Ohta K., et al., Chem. Pharm. Bull., 46, 684-686, 1998). It was a new sulfolipid compound that belonged in the class of sulfoquinovosyldiacylglycerol. The biochemical properties have been investigated here. The compound, temporarily designated KM043, potently inhibited the activities of mammalian DNA polymerase alpha(pol. alpha) and DNA polymerase beta(pol. beta) and terminal deoxynucleotidyl transferase (TdT), and moderately, human immunodeficiency virus reverse transcriptase (HIV-RT). KM043 dose-dependently inhibited their activities, and each of their IC50 values was 0.25 microM for pol. alpha, 0.38 microM for TdT, 3.6 microM for pol. beta, or 11.2 microM for HIV-RT, and almost complete inhibition of each was achieved at 1.0 to 2.0 microM for pol. alpha and TdT, 7.5 microM for pol. beta and about 30 microM for HIV-RT. However, the compound did not influence the activities of prokaryotic DNA polymerases such as E. coli DNA polymerase I, and DNA metabolic enzymes like DNase 1. Inhibition of pol. alpha or beta by KM043 was non-competitive with both the DNA template and the substrate deoxythymidine 5'-triphosphate (dTTP). KM043 was weakly cytotoxic to cultured HeLa-S3 cells, and the IC50 value was 80 microM. KM043 could synergistically enhance the cytocidal effect of an anti-cancer chemotherapy agent, bleomycin. In the presence of 50 microM KM043, the effect ratio of (bleomycin plus KM043)/(bleomysin only) decreased from 0.76 to 0.22.