Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase alpha-primase

Human cell extracts efficiently support replication of simian virus 40 (SV40) DNA in vitro, while mouse cell extracts do not. Since human DNA polymerase alpha-primase is the major species-specific factor, we set out to determine the subunit(s) of DNA polymerase alpha-primase required for this species specificity. Recombinant human, mouse, and hybrid human-mouse DNA polymerase alpha-primase complexes were expressed with baculovirus vectors and purified. All of the recombinant DNA polymerase alpha-primases showed enzymatic activity and efficiently synthesized the complementary strand on an M13 single-stranded DNA template. The human DNA polymerase alpha-primase (four subunits [HHHH]) and the hybrid DNA polymerase alpha-primase HHMM (two human subunits and two mouse subunits), containing human p180 and p68 and mouse primase, initiated SV40 DNA replication in a purified system. The human and the HHMM complex efficiently replicated SV40 DNA in mouse extracts from which DNA polymerase alpha-primase was deleted, while MMMM and the MMHH complex did not. To determine whether the human p180 or p68 subunit was required for SV40 DNA replication, hybrid complexes containing only one human subunit, p180 or p68, together with three mouse subunits (HMMM and MHMM) or three human subunits and one mouse subunit (MHHH and HMHH) were tested for SV40 DNA replication activity. The hybrid complexes HMMM and HMHH synthesized oligoribonucleotides in the SV40 initiation assay with purified proteins and replicated SV40 DNA in depleted mouse extracts. In contrast, the hybrid complexes containing mouse p180 were inactive in both assays. We conclude that the human p180 subunit determines host-specific replication of SV40 DNA in vitro.     

Phosphorylation of DNA polymerase alpha-primase by cyclin A-dependent kinases regulates initiation of DNA replication in vitro

DNA polymerase alpha-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In this study, we investigated the regulation of DNA replication by phosphorylation of DNA polymerase alpha-primase. The p180 and the p68 subunits of DNA polymerase alpha-primase were phosphorylated using Cyclin A-, B- and E- dependent kinases. This phosphorylation did not influence its DNA polymerase activity on activated DNA, but slightly stimulated primase activity using poly(dT) single-stranded DNA (ssDNA) without changing the product length of primers. In contrast, site-specific initiation of replication on plasmid DNA containing the SV40 origin is affected: Cyclin A-Cdk2 and Cyclin A-Cdc2 inhibited initiation of SV40 DNA replication in vitro, Cyclin B-Cdc2 had no effect and Cyclin E-Cdk2 stimulated the initiation reaction. DNA polymerase alpha-primase that was pre-phosphorylated by Cyclin A-Cdk2 was completely unable to initiate the SV40 DNA replication in vitro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, while Cyclin E-Cdk2-treated DNA polymerase alpha-primase remained fully active in the initiation reaction.     

Interrelationships between strains of Salmonella enteritidis belonging to phage types 4, 7, 7a, 8, 13, 13a, 23, 24 and 30

Cyclin A is a nuclear protein which is part of a kinase complex with either p34cdc2 or p33cdk2. Cyclin A is required in higher eukaryotic cells at the G1/S and the G2/M transitions. To examine the relationship between cyclin A and DNA replication, we simultaneously labeled exponentially growing HeLa cells for the distribution of cyclin A and proliferating cell nuclear antigen (PCNA). We have now demonstrated, by means of immunoelectron microscopy, that cyclin A is located at the sites of DNA replication visualized by both BrdU and PCNA labeling. Thus cyclin A may play a significant role in the phosphorylation of proteins at or near the sites of DNA replication.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Inhibition of antigen-induced muscle contractions by inhibitors of thromboxane pathway in rat small intestine

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human breast carcinoma cells by prostaglandin A2

Prostaglandin A2 (PGA2) potently inhibits cell proliferation and suppresses tumor growth in vivo, but little is known regarding the molecular mechanisms mediating these effects. Here we demonstrate that treatment of breast carcinoma MCF-7 cells with PGA2 leads to G1 arrest associated with a dramatic decrease in the levels of cyclin D1 and cyclin-dependent kinase 4 (cdk4) and accompanied by an increase in the expression of p21. We further show that these effects occur independent of cellular p53 status. The decline in cyclin D and cdk4 protein levels is correlated with loss in cdk4 kinase activity, cdk2 activity is also significantly inhibited in PGA2-treated cells, an effect closely associated with the upregulation of p21. Immunoprecipitation experiments verified that p21 was indeed complexed with cdk2 in PGA2-treated cells. Additional experiments with synchronized MCF-7 cultures stimulated with serum revealed that treatment with PGA2 prevents the progression of cells from G1 to S. Accordingly, the kinase activity associated with cdk4, cyclin E, and cdk2 immunocomplexes, which normally increases following serum addition, was unchanged in PGA2-treated cells. Furthermore, the retinoblastoma protein (Rb), a substrate of cdk4 and cdk2 whose phosphorylation is necessary for cell cycle progression, remains underphosphorylated in PGA2-treated serum-stimulated cells. These findings indicate that PGA2 exerts its growth-inhibitory effects through modulation of the expression and/or activity of several key G1 regulatory proteins. Our results highlight the chemotherapeutic potential of PGA2, particularly for suppressing growth of tumors lacking p53 function.     

Functional interactions between SV40 T antigen and other replication proteins at the replication fork

The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen.     

Rapid and selective depletion of CD4+ T lymphocytes and preferential loss of memory cells on interaction of mononuclear cells with HIV-1 glycoprotein-expressing cells

Apigenin is a plant flavonoid that has been shown to significantly inhibit ultraviolet-induced mouse skin tumorigenesis when applied topically and may be an alternative sunscreen agent for humans. A long-term goal of our laboratory is to elucidate the molecular mechanism or mechanism by which apigenin inhibits skin tumorigenesis. In a previous publication, we characterized the mechanism by which apigenin induced G2/M arrest in keratinocytes. More recent studies in our laboratory have provided evidence that apigenin can induce G1 arrest in addition to arresting cells at G2/M. Here we describe the mechanism of the apigenin-induced G1 arrest in human diploid fibroblasts (HDF). Treatment of asynchronous HDF for 24 h with 10-50 microM apigenin resulted in dose-dependent cell-cycle arrest at both the G0/G1 and G2/M phases as measured by flow cytometry. The G0/G1 arrest was more clearly defined by using HDF that were synchronized in G0 and then released from quiescence by replating at subconfluent densities in medium containing 10-70 microM apigenin. The cells were analyzed for cell-cycle progression or cyclin D1 expression 24 h later. A dose of apigenin as low as 10 microM reduced the percentage of cells in S phase by 20% compared with control cultures treated with solvent alone. Western blot analysis of apigenin-treated HDF indicated that cyclin D1 was expressed at higher levels than in untreated cells, which signifies that they were arrested in G1 phase rather than in a G0 quiescent state. The G1 arrest was further studied by cyclin-dependent kinase 2 (cdk2) immune complex-kinase assays of apigenin-treated asynchronous HDF, which demonstrated a dose-dependent inhibition of cdk2 by apigenin. Inhibition of cdk2 kinase activity in apigenin-treated cells was associated with the accumulation of the hypophosphorylated form of the retinoblastoma (Rb) protein as measured by western blot analysis. The cdk inhibitor p21/WAF1 was also induced in a dose-dependent manner, with a 22-fold induction of p21/WAF1 in 70 microM apigenin-treated cells. In conclusion, apigenin treatment produced a G1 cell-cycle arrest by inhibiting cdk2 kinase activity and the phosphorylation of Rb and inducing the cdk inhibitor p21/WAF1, all of which may mediate its chemopreventive activities in vivo. To our knowledge this is the first report of a chemopreventive agent inducing p21/WAF1, a known downstream effector of the p53 tumor suppressor protein.     

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.     

Characterization of the in vitro reconstituted cyclin A or B1-dependent cdk2 and cdc2 kinase activities

Human cyclins A and B1 were assembled with the cdk2 or cdc2 protein to reconstitute their respective kinase activities in vitro. Both cyclins complemented either cdk2 or cdc2, yielding kinase activities that supported the phosphorylation of histone H1. Activation of cdk2-catalyzed H1 kinase activity by cyclin A required a 10-min preincubation of the two components, whereas cdc2 kinase supported phosphate incorporation without a detectable time lag upon the addition of cyclin B1, suggesting a slower association rate of cdk2 with cyclin A compared with cdc2 and cyclin B1. Both cdk2 and cyclin A, as well as cdc2 and cyclin B1, formed stable complexes in the absence of ATP and substrate that could be isolated after glycerol gradient centrifugation. Incubation of the isolated complexes with ATP and histone H1 supported the phosphorylation of the substrate. Cyclin A-activated cdk2 or cdc2 phosphorylated p107, a pRB-related cellular protein, 10 times more effectively than the cyclin B1-complexed kinases. This was most likely due to a direct association of cyclin A with p107 (Ewen, M. E., Faha, B., Harlow, E., and Livingston, D. (1992) Science 255, 85-87; Faha, B., Ewen, M. E., Tsai, L.-H., Livingston, D., and Harlow, E. (1992) Science 255, 87-90). The reconstituted cdc2-cyclin B1 complex incorporated 4-5-fold more phosphate into the p34 subunit of the three-subunit (p70, p34, and p14) human single-stranded DNA-binding protein (also called RP-A), a DNA replication and DNA repair factor, than cdc2-cyclin A. No detectable phosphorylation of the p34 protein was observed with cdk2 complexed with either cyclin B1 or A. These data indicate that both cyclins as well as the catalytic subunits are important factors in controlling the rate of phosphorylation of a given substrate. The cyclin-activated cdc2 family kinases may target their cellular substrates through cyclin-mediated protein-protein interactions.     

Sustained accumulation of the mitotic cyclins and tyrosine-phosphorylated p34cdc2 in human G1-S-arrested cancer cells but not untransformed cells

Coupling mitosis to the completion of DNA replication in cycling embryonic extracts from Xenopus eggs appears to rely on blocking the activation of the tyrosine-phosphorylated p34cdc2/cyclin B, which continues to build up when S phase is inhibited by adding unreplicated DNA (Smythe, C., and Newport, J. W., Cell, 68: 787-797, 1992). We show here that a similar mechanism might be operative in human tumor-derived cells, which, during a thymidine-aphidicolin block, stop progressing through S phase and thereby fail to undergo mitosis. Under such conditions, indeed, cancer cells do continue to accumulate cyclin A, cyclin B1, and tyrosine-phosphorylated p34cdc2 to supranormal levels, a phenomenon that does not occur in untransformed, nonimmortalized human fibroblasts. Thus, in human cancer cells, the onset of active accumulation of cyclin A and cyclin B1 can be uncoupled from transit through the G1-S and S-G2 borders, respectively, and, as in simple embryonic cell cycles, the coupling of mitosis to the completion of S phase presumably relies, at least in part, on the prevention of premature activation of the tyrosine-phosphorylated p34cdc2/cyclin B1 complex.     

Proteolysis and tyrosine phosphorylation of p34cdc2/cyclin B. The role of MCM2 and initiation of DNA replication to allow tyrosine phosphorylation of p34cdc2

Previously, it has been shown that Aspergillus cells lacking the function of nimQ and the anaphase-promoting complex (APC) component bimEAPC1 enter mitosis without replicating DNA. Here nimQ is shown to encode an MCM2 homologue. Although mutation of nimQMCM2 inhibits initiation of DNA replication, a few cells do enter mitosis. Cells arrested at G1/S by lack of nimQMCM2 contain p34(cdc2)/cyclin B, but p34(cdc2) remains tyrosine dephosphorylated, even after DNA damage. However, arrest of DNA replication using hydroxyurea followed by inactivation of nimQMCM2 and bimEAPC1 does not abrogate the S phase arrest checkpoint over mitosis. nimQMCM2, likely via initiation of DNA replication, is therefore required to trigger tyrosine phosphorylation of p34(cdc2) during the G1 to S transition, which may occur by inactivation of nimTcdc25. Cells lacking both nimQMCM2 and bimEAPC1 are deficient in the S phase arrest checkpoint over mitosis because they lack both tyrosine phosphorylation of p34(cdc2) and the function of bimEAPC1. Initiation of DNA replication, which requires nimQMCM2, is apparently critical to switch mitotic regulation from the APC to include tyrosine phosphorylation of p34(cdc2) at G1/S. We also show that cells arrested at G1/S due to lack of nimQMCM2 continue to replicate spindle pole bodies in the absence of DNA replication and can undergo anaphase in the absence of APC function.     

Photodynamic therapy results in induction of WAF1/CIP1/P21 leading to cell cycle arrest and apoptosis

Photodynamic therapy (PDT) is a promising new modality that utilizes a combination of a photosensitizing chemical and visible light for the management of a variety of solid malignancies. The mechanism of PDT-mediated cell killing is not well defined. We investigated the involvement of cell cycle regulatory events during silicon phthalocyanine (Pc4)-PDT-mediated apoptosis in human epidermoid carcinoma cells A431. PDT resulted in apoptosis, inhibition of cell growth, and G0-G1 phase arrest of the cell cycle, in a time-dependent fashion. Western blot analysis revealed that PDT results in an induction of the cyclin kinase inhibitor WAF1/CIP1/p21, and a down-regulation of cyclin D1 and cyclin E, and their catalytic subunits cyclin-dependent kinase (cdk) 2 and cdk6. The treatment also resulted in a decrease in kinase activities associated with all the cdks and cyclins examined. PDT also resulted in (i) an increase in the binding of cyclin D1 and cdk6 toward WAF1/CIP1/p21, and (ii) a decrease in the binding of cyclin D1 toward cdk2 and cdk6. The binding of cyclin E and cdk2 toward WAF1/CIP1/p21, and of cyclin E toward cdk2 did not change by the treatment. These data suggest that PDT-mediated induction of WAF1/CIP1/p21 results in an imposition of artificial checkpoint at G1 --> S transition thereby resulting in an arrest of cells in G0-G1 phase of the cell cycle through inhibition in the cdk2, cdk6, cyclin D1, and cyclin E. We suggest that this arrest is an irreversible process and the cells, unable to repair the damages, ultimately undergo apoptosis.     

Modulation of p27(Kip1) levels by the cyclin encoded by Kaposi's sarcoma-associated herpesvirus

DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposi's sarcoma-associated herpesvirus) which is able to resist the actions of p16(Ink4a), p21(Cip1) and p27(Kip1) cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27(Kip1). We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin-cdk2 complexes but not cyclin D1-cdk6. Included amongst these substrates is p27(Kip1) which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27(Kip1) downregulation, this being consistent with previous studies indicating that phosphorylation of p27(Kip1) on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27(Kip1) in which Thr187 is mutated to non-phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27(Kip1)-imposed G1 arrest by facilitating phosphorylation and downregulation of p27(Kip1) to enable activation of endogenous cyclin-cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus-encoded cyclin.     

The CDK inhibitor, p27Kip1, is required for IL-4 regulation of astrocyte proliferation

IL-4 is a pleiotrophic cytokine that has been shown to affect cells of the central nervous system. We have demonstrated that IL-4 inhibits DNA synthesis and proliferation in human astroglia expressing IL-4 receptors. In this study, we sought to identify mechanisms that could account for the antimitogenic effects of IL-4. Epidermal growth factor (EGF)-stimulated human astroglia were arrested in G1 phase by IL-4, even though IL-4 stimulated levels of the G1 cyclins, D1 and E. Histone H1 kinase activity of cdk2 immunoprecipitates, however, was sharply reduced by IL-4; impairment of kinase activity was also evident in cyclin E immunoprecipitates, which contained evidence of hypophosphorylated (inactive) cdk2 product. Reduced cyclin E-associated cdk2 activity was not due to impaired cyclin-dependent kinase-activating kinase (CAK) activity, which was unaffected by IL-4. Inactive cyclin E/cdk2 complexes from IL-4 + EGF-treated cells contained, however, strikingly elevated p27Kip1 cdk inhibitor. Elevated p27 was also detectable in whole cell lysates after 24 and 48 h of IL-4 treatment; by 72 h, p27 was no longer elevated. Pretreatment with antisense but not mismatch p27 oligonucleotides attenuated the inhibitory effects of IL-4 on DNA synthesis and histone kinase activity of cyclin E/cdk2 complexes. Antisense p27 also abrogated IL-4-mediated elevation of p27 in whole cell lysates and cyclin E/cdk2 complexes. These findings demonstrate that IL-4 regulates the cell cycle machinery of astroglial cells via a p27Kip1 braking mechanism.     

Evidence for a Cdc6p-independent mitotic resetting event involving DNA polymerase alpha

Eukaryotic DNA replication is limited to once per cell cycle because cyclin-dependent kinases (cdks), which are required to fire origins, also prevent re-replication. Components of the replication apparatus, therefore, are 'reset' by cdk inactivation at the end of mitosis. In budding yeast, assembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) at origins can only occur during G1 because it is blocked by cdk1 (Cdc28) together with B cyclins (Clbs). Here we describe a second, separate process which is also blocked by Cdc28/Clb kinase and, therefore, can only occur during G1; the recruitment of DNA polymerase alpha-primase (pol alpha) to chromatin. The recruitment of pol alpha to chromatin during G1 is independent of pre-RC formation since it can occur in the absence of Cdc6 protein. Paradoxically, overproduction of Cdc6p can drive both dephosphorylation and chromatin association of pol alpha. Overproduction of a mutant in which the N-terminus of Cdc6 has been deleted is unable to drive pol alpha chromatin binding. Since this mutant is still competent for pre-RC formation and DNA replication, we suggest that Cdc6p overproduction resets pol alpha chromatin binding by a mechanism which is independent of that used in pre-RC assembly.     

TGF-beta mediated G1 arrest in a human melanoma cell line lacking p15INK4B: evidence for cooperation between p21Cip1/WAF1 and p27Kip1

We have studied TGF-beta mediated G1 arrest in WM35, an early stage human melanoma cell line. These cells have lost p15INK4B expression through loss of one chromosome 9 and rearrangement of the other. In asynchronously growing WM35, TGF-beta caused reductions in cyclin D1, cyclin A and cdk4 proteins and their associated kinase activities and an increase in both p21Cip1/WAF1 and p27Kip1. These findings were confirmed in cells released from quiescence in the presence of TGF-beta, in which TGF-beta inhibited or delayed the reduction in the cdk inhibitors that normally occurs in late G1. In contrast to observations in other cell types, there was an increased association of both p21Cip1/WAF1 and p27Kip1 with cyclin D1/cdk4 and with cyclin E/cdk2 during TGF-beta mediated arrest of asynchronously growing cells. Upregulation of p21Cip1/WAF1 preceded that of p27Kip1. Furthermore, p21Cip1/WAF1 and p27Kip1 were not present in the same cdk complexes but bound distinct populations of target cdk molecules. Both p21Cip1/WAF1 and p27Kip1 immunoprecipitates from asynchronously growing cells contained active kinase complexes. These KIP-associated kinase activities were reduced in TGF-beta arrested cells. It has been proposed that in TGF-beta arrested epithelial cells, up-regulation of p15INK4B and of p15INK4B binding to cdk4 serves to destabilize the association of p27Kip1 with cyclin D1/cdk4, promoting p27Kip1 binding and inhibition of cyclin E/cdk2. Our findings demonstrate that this is not a universal mechanism of G1 arrest by TGF-beta. In TGF-beta arrested WM35, which lack p15INK4B, the increased p21Cip1/WAF1 may serve a similar function to that of p15INK4B: initiating kinase inhibition and providing an additional mechanism to supplement the effect of p27Kip1 on G1 cyclin/cdks.     

Identification of a p130 domain mediating interactions with cyclin A/cdk 2 and cyclin E/cdk 2 complexes

P130 shares structural and functional homology with pRb and p107. One property common to p107 and p130, but not to pRb, is the ability to stably interact with cyclin A/cdk2 and cyclin E/cdk2 complexes in vitro and in vivo. Using GST-p130 fusion proteins representing various regions of p130, baculovirus-produced cyclin A/cdk2 and cyclin E/cdk2 complexes were found to interact with residues within a part of p130 known as the spacer region. Cyclin E was able to bind the p130 spacer region in the presence or absence of cdk2 whereas cyclin A binding was dependent upon the presence of cdk2. The smallest p130 fusion protein sufficient to interact with cyclin A/cdk2 or cyclin E/cdk2 complexes contained p130 amino acids 652-698 and deletion of p130 amino acids 680-682 abolished binding to both of the cyclin/cdk2 complexes. When overexpressed in C33A cells, a p130 mutant containing a deletion of amino acids 620-697 was unable to form complexes with either cyclin A or cyclin E. This p130 mutant was at least as active as wild type p130 in suppressing the growth of G418 resistant colonies when overexpressed in C33A or SAOS-2 cells.     

Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit

DNA polymerase alpha-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase alpha-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.