Phosphorylation: the molecular switch of double-strand break repair.

Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

Smad 3 may regulate follicular growth in the mouse ovary.

Although Smad 3 is known to serve as a signaling intermediate for the transforming growth factor beta (TGFbeta) family in nonreproductive tissues, its role in the ovary is unknown. Thus, we used a recently generated Smad 3-deficient (Smad 3-/-) mouse model to test the hypothesis that Smad 3 alters female fertility and regulates the growth of ovarian follicles from the primordial stage to the antral stage. In addition, we tested whether Smad 3 affects the levels of proteins that control apoptosis, survival, and proliferation in the ovarian follicle. To test this hypothesis, breeding studies were conducted using Smad 3-/- and wild-type mice. In addition, ovaries were collected from Smad 3-/- and wild-type mice on Postnatal Days 2-90. One ovary from each animal was used to estimate the total number of primordial, primary, and antral follicles. The other ovary was used for immunohistochemical analysis of selected members of the B-cell lymphoma/leukemia-2 family of protooncogenes (Bax, Bcl-2, Bcl-x), proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (Cdk-2). The results indicate that Smad 3-/- mice have reduced fertility compared with wild type mice. The results also indicate that Smad 3 may not affect the size of the primordial follicle pool at birth, but it may regulate growth of primordial follicles to the antral stage. Further, the results indicate that Smad 3 may regulate the expression of Bax and Bcl-2, but not Bcl-x, Cdk-2, and PCNA. Collectively, these data suggest that Smad 3 may play an important role in the regulation of ovarian follicle growth and female fertility.

Ara-C differentially affects multiprotein forms of human cell DNA polymerase.

PURPOSE: The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has proven to be one of the most effective agents available for the treatment of acute leukemia although the precise mechanism by which ara-C induces cytotoxicity remains unclear. Our laboratory has previously isolated from human cells a DNA replication complex, termed the DNA synthesome, which is fully competent to orchestrate, in vitro, all of the reactions required to efficiently and faithfully replicate DNA. Using this system and the active metabolite of ara-C, ara-CTP, we demonstrated that the human DNA synthesome can efficiently incorporate ara-CTP into internucleotide positions of newly replicated DNA in vitro mimicking results obtained using intact cells and isolated nuclei. We then hypothesized that DNA polymerase auxiliary proteins, present within the DNA synthesome, may aid in incorporating this nucleotide analog into DNA. METHODS: To test this hypothesis, we utilized three distinct multiprotein complexes each of which contained human DNA polymerase alpha and examined with standard in vitro polymerase assays the effectiveness of ara-C in inhibiting various aspects of their polymerase function. RESULTS AND CONCLUSION: These polymerase-mediated elongation assays, which included ara-CTP- or ara-C-containing primers in the reaction mixture, showed that the rate of DNA elongation in the presence of ara-CTP was significantly enhanced when the DNA polymerase was associated with its auxiliary proteins, and that the elongation resulted in the formation of internucleotide ara-CMP. Nevertheless, the enhanced activities resulting from the association of these auxiliary proteins with polymerase alpha did not fully account for the remarkable efficiency with which the DNA synthesome incorporated ara-C into internucleotide positions during DNA replication.

Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP).

The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.

Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff.

Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.

Ara-C affects formation of cancer cell DNA synthesome replication intermediates.

PURPOSE: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. METHODS: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta). RESULTS: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug. CONCLUSIONS: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

A neutralizing antibody against human DNA polymerase epsilon inhibits cellular but not SV40 DNA replication.

The contribution of human DNA polymerase epsilon to nuclear DNA replication was studied. Antibody K18 that specifically inhibits DNA polymerase activity of human DNA polymerase epsilon in vitro significantly inhibits DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei. The capability of this neutralizing antibody to inhibit DNA synthesis in cells is comparable to that of monoclonal antibody SJK-132-20 against DNA polymerase alpha. Contrary to the antibody against DNA polymerase alpha, antibody K18 against DNA polymerase epsilon did not inhibit SV40 DNA replication in vitro. These results indicate that DNA polymerase epsilon plays a role in replicative DNA synthesis in proliferating human cells like DNA polymerase alpha, and that this role for DNA polymerase epsilon cannot be modeled by SV40 DNA replication.

Human breast cancer cells contain an error-prone DNA replication apparatus.

The mechanisms responsible for creating genetic errors and genomic instability in cancer cells have not been fully defined. Recently, it has been shown that human cells contain a highly organized complex of proteins, termed the DNA synthesome, that is fully competent to carry out all phases of SV40 in vitro DNA replication (J. M. Coll et al, Oncol. Res., 8: 435-447, 1996; L. H. Malkas et al., Biochemistry, 29: 6362-6374, 1990; Y. Wu et al., J. Cell. Biochem., 54: 32-46, 1994; N. Applegren et al., J. Cell. Biochem., 54: 32-46, 1994). DNA replication fidelity analyses of the DNA synthesome derived from malignant and nonmalignant human breast cells demonstrate that the malignant cell synthesome is mutagenic. The decrease in tumor cell replication fidelity was not due to an increased proliferative capacity of the tumor cells or an increase in the synthetic activity of their DNA synthesome. The ratios of insertions, deletions, and mismatches created by the synthesome from malignant and nonmalignant breast cells were essentially identical, despite the greater overall number of mutations made by the breast cancer cell synthesome. These data define, for the first time, a mechanism unique to cancer cells that contributes to the observed increase in genetic mutation in cancer cells.

A unique form of proliferating cell nuclear antigen is present in malignant breast cells.

Despite extensive research efforts to identify unique molecular alterations in breast cancer, to date, no characteristic has emerged that correlates exclusively with malignancy. Recently, it has been shown that the multiprotein DNA replication complex (synthesome) from breast cancer cells has a significantly decreased replication fidelity compared to that of nonmalignant breast cells. Proliferating cell nuclear antigen (PCNA) functions in DNA replication and DNA repair and is a component of the synthesome. Using two-dimensional PAGE analysis, we have identified a novel form of PCNA in malignant breast cells. This unique form is not the result of a genetic alteration, as demonstrated by DNA sequence analysis of the PCNA gene from malignant and nonmalignant breast cells. This novel form is most likely the result of an alteration in the post-translational modification of PCNA in malignant breast cells. These findings are significant in that it is now possible to link changes in the fidelity of DNA replication with a specific alteration of a component of the DNA synthetic apparatus of breast cancer cells. The novel form of PCNA may prove to be a new signature for malignant breast cells.

Regulation of the expression or recruitment of components of the DNA synthesome by poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.

Anthracycline antibiotic blockade of SV40 T antigen helicase action.

We previously showed that anthracycline antibiotics potently block SV40 large T antigen helicase; in the present study, we describe the kinetics and the structure-activity characteristics of this process. The concentration vs effect data for helicase blockade were fitted by the Hill equation to yield nearly parallel log-concentration effect curves for a series of active anthracycline antibiotics. The effective concentration for 50% helicase blockade (EC50) values ranged from 0.34 microM for daunorubicin to 40.8 microM for 3'-deaminodaunorubicin. Clinically inactive 3'-N-acyl anthracyclines produced no blockade. The Hill constants for the blockade ranged from 1.1 to 1.6 for the entire series of active anthracyclines, indicating no positive cooperativity and suggesting that a single molecule of bound drug is sufficient to block helicase action. The EC50 values for several clinically effective anthracyclines showed a relationship to the average DNA binding constants for these drugs, and Lineweaver-Burk analysis of the blockade kinetics indicated non-competitive inhibition. The kinetics of the blockade indicated that the anthracycline, DNA, and helicase form a ternary complex that is irreversible under the reaction conditions. This mechanism may be central to the cytotoxic and anti-cancer activities of anthracycline antibiotics and may be useful in understanding the enzymatic mechanism of DNA helicase action.

DNA replication machinery of the mammalian cell.

The process of DNA replication in mammalian cells is highly complex and has several unique features that distinguish it from simpler prokaryotic systems. The study of mammalian DNA replication lagged behind that of prokaryotes for many years. This was because of the lack of a reliable and efficient mammalian cell-based in vitro DNA replication system. In 1984, the first mammalian-based DNA replication system that initiated DNA synthesis successfully in vitro was developed. The employment of the mammalian in vitro DNA replication system has led to the identification of several DNA replication proteins. This article describes the current knowledge regarding the proteins mediating mammalian DNA replication, as well as how they are proposed to function during DNA synthesis. There is also a discussion of the role the mammalian cell nuclear architecture plays in DNA replication. The evidence for the existence of an organized DNA replication machine in mammalian cells is also presented.

Mercuric ion inhibits the activity and fidelity of the human cell DNA synthesome.

Mercuric ion is cytotoxic and mutagenic to cells; however, the mechanisms of mercuric ion-induced cytotoxicity are not well understood. Numerous studies have suggested that these effects may be due in part to the alteration and inhibition of a variety of cellular processes including DNA replication, DNA repair, RNA transcription, and protein synthesis. Studies utilizing whole cells to examine these activities are not able to specifically identify the precise mechanism or site of the effect. Other studies carried out using whole cell extracts and variously purified DNA polymerases are not able to adequately represent the highly ordered environment in which DNA replication occurs in the intact cell. We report here, for the first time, the use of an intact human cell multiprotein complex (which we have termed the DNA synthesome) to carry out full-length DNA replication and DNA synthesis in the presence of Hg2+ ion in vitro. In this study we report that DNA replication and DNA polymerase activity, as well as DNA replication fidelity of the human cell DNA synthesome, are specifically inhibited by physiologically attainable concentrations of mercuric ion.

The isolation of a DNA synthesome from human leukemia cells.

In this report we describe, for the first time, the purification and characterization of a replication-competent multiprotein form of DNA polymerase (designated the DNA synthesome) from the human leukemia cell line (HL-60) using a series of centrifugation, ion-exchange chromatography and velocity sedimentation steps. The proteins and enzymatic activities thus far identified to co-purify with the leukemia cell DNA synthesome include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), and DNA topoisomerases I and II. We have demonstrated that the DNA synthesome is fully competent to replicate simian virus 40 (SV40) replication origin containing DNA in vitro in the presence of the viral large T-antigen. This result implies that all of the cellular activities required for large T-antigen-dependent in vitro SV40 DNA synthesis are present in the isolated human leukemia cell DNA synthesome. Since SV40 is extensively dependent on the host cell's DNA synthetic machinery for its own DNA replication, our results indicate that the isolated leukemia cell DNA synthesome may play a role not only in viral DNA synthesis but also in human leukemia cell DNA replication. We recently proposed a model to represent the DNA synthesome that was isolated from HeLa and murine cells. Our data indicate that the organization of the DNA synthesome from HL-60 cells also fits this proposed model. The purified DNA synthesome will not only allow the further study of the molecular mechanisms required to carry out human leukemia cell DNA replication, but may also provide a tool for eventually dissecting some of the regulatory controls of the cell's DNA synthetic machinery.

Mammalian cell DNA replication.

The precise mechanisms involved in the regulation of the mammalian cell DNA-synthesizing machinery are poorly understood. In vitro DNA replication systems, in particular the employment of the simian virus 40 (SV40)-based cell-free DNA replication system, has identified several mammalian enzymes and proteins required for DNA synthesis. Although these proteins have been identified as playing a role in DNA replication, their functional organization allowing for the efficient replication of DNA has not been well defined. This review describes the proteins that have currently been defined as having a role in mammalian DNA replication and their proposed mechanisms of action. How these proteins may organize themselves to form multiprotein complexes, or larger DNA replication factories, allowing for efficient chromosomal DNA synthesis is discussed. In addition, the cell cycle regulation of mammalian DNA synthesis and the current status concerning mammalian DNA replication origins is described.

Mapping specific protein-protein interactions within the core component of the breast cell DNA synthesome.

We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the DNA synthesome, from human breast cancer cells and biopsied human breast tumor tissue. The purified DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell DNA synthesome, in which DNA polymerases alpha, delta, and epsilon, DNA primase, and replication factor C (RF-C) represent members of the core component, or tightly associated, proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these proteins. We report here that poly(ADP-ribose)polymerase (PARP) and DNA ligase 1 are also members of the breast cell DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the protein-protein interactions between several members of the core component of the DNA synthesome. Consistent with our proposed model for the breast cell DNA synthesome, our data indicate that DNA polymerases alpha and delta, DNA primase, RF-C, as well as proliferating cell nuclear antigen (PCNA), tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only PCNA or DNA polymerase alpha. The association of PARP with the synthesome core suggests that this protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell DNA. To our knowledge this is the first report ever to describe the close physical association of polypeptides constituting the intact human breast cell DNA replication apparatus.

The biochemical status of the DNA synthesome can distinguish between permanent and temporary cell growth arrest.

We previously identified and characterized the human leukemia (HL-60) cell DNA synthetic machinery as a multiprotein form of DNA polymerase, which was designated the DNA synthesome. This multiprotein replication complex contains DNA polymerases alpha and delta, primase, replication factor C, replication protein A, helicase, poly(ADPribose) polymerase, proliferating cell nuclear antigen, DNA ligase I, and topoisomerases I and II. Recently, the HeLa cell-derived DNA synthesome was identified as a discrete high molecular weight protein band in native polyacrylamide gels. Here, we report our findings regarding the change in the organizational status of the DNA synthesome when HL-60 cells undergo either terminal differentiation or temporary G1 growth arrest. We observed that the HL-60 cell DNA synthesome also migrates as a discrete high molecular weight protein band in nondenaturing polyacrylamide gels. This high molecular weight protein band was present in nuclei derived from both actively cycling cells and aphidicolin-arrested cells but was absent in TPA-induced terminally differentiated cells. We also found that DNA polymerase delta, replication factor C, and proliferating cell nuclear antigen are absent in cells that are induced to differentiate in response to 12-O-tetradecanoyl phorbol-13-acetate treatment but are present in actively cycling cells. The level of replication protein A in differentiated cells was similar to that of cycling cells, whereas the level of annexin I, a cytoskeleton protein, is higher in differentiated cells than it is in actively cycling cells. We conclude that the DNA synthesome remains integrated and inactive in temporarily growth-arrested cells but is disassembled in differentiated cells. Furthermore, we conclude that disassembly of the organized replication complex is a specific cellular event in the process of permanent cell cycle exit and that the process leading to disassembly may be regulated, in part, at the level of gene transcription.

Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity.

Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin-specific large T-antigen-dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase alpha and DNA polymerase delta, respectively. To detect epitopes possibly unexposed within the native multiprotein complexes, blots were also analyzed following denaturation in situ following treatment with detergent and reducing agent. The epitope or access to the epitope recognized by the monoclonal antibody against DNA polymerase alpha was destroyed by exposure of the blots to denaturing conditions. In contrast, an epitope present on a very large complex of approximately 1000 kDa was recognized by a monoclonal antibody against proliferating cell nuclear antigen only following treatment of the native immunoblots with denaturing agents. Identification of these complexes will allow their further purification, characterization, and elucidation of their role in the replication of DNA.

The expression of poly(ADP-ribose) polymerase during differentiation-linked DNA replication reveals that it is a component of the multiprotein DNA replication complex.

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.