Effect of anti-cruciform DNA monoclonal antibodies on DNA replication.

To study the possible involvement of DNA cruciforms in the initiation of DNA replication, we used two monoclonal antibodies, 2D3 and 4B4, with anti-cruciform DNA specificity. Synchronized CV-1 cells were released into S phase for hourly intervals up to 6 h and permeabilized in the presence of monoclonal antibodies, under conditions that allow limited DNA replication. Exposure of the permeabilized cells to 2D3 or 4B4 resulted in a 2- to 6-fold enhancement of incorporation of labeled precursor nucleotide over the 6 h period. Approximately 50% of the enhanced synthesis was sensitive to aphidicolin, and the enhancing effect of 2D3 was abolished by absorption with immunobead anti-mouse immunoglobulin. Dot-blot hybridization analyses of DNA isolated from anti-cruciform antibody treatment groups showed a similar 2- to 11-fold increase in the relative copy number of low copy probes. In contrast, exposure of the permeabilized cells to a monoclonal antibody directed against Z-DNA and B-DNA had no significant effect on DNA synthesis. The results suggest that cruciforms are present in replicating DNA and that they are recognized and stabilized by the monoclonal antibodies.     

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase α revealed the intranuclear localization of DNA polymerase α in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the α-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase a appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase a by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.     

Calmodulin regulates DNA polymerase alpha activity during proliferative activation of NRK cells.

When Normal Rat Kidney cells are allowed to reenter the cell cycle after quiescence they start to replicate DNA around 12 h, reaching a maximum at 20 h. Activation of DNA polymerase alpha parallels the increase in DNA synthesis. The addition of two different anti-calmodulin drugs, trifluoroperazine (7.5 microM) or W13 (10 micrograms/ml), to the media at 4 h after proliferative activation, inhibits DNA synthesis by 55% and 80%, respectively. The blockade of calmodulin produced by trifluoroperazine allows the cells to progress through G1 phase but stops progression through S phase as determined by 5-Bromo deoxyuridine labeling. Both anti-calmodulin drugs also inhibit by more than 50% the increase in DNA polymerase alpha activity observed at 20 h. These results indicate that a calmodulin-dependent event, essential for the activation of DNA polymerase alpha and subsequently for DNA replication, is produced during G1. Therefore, the control of DNA polymerase alpha activation is one of the ways by which calmodulin is regulating the progression of NRK cells through S phase.     

Activity levels of mouse DNA polymerase alpha-primase complex (DNA replicase) and DNA polymerase alpha, free from primase activity in synchronized cells, and a comparison of their catalytic properties

To asses the possible roles of the two active forms of mouse DNA polymerase alpha: primase--DNA-polymerase alpha complex (DNA replicase) and DNA polymerase alpha free from primase activity (7.3S polymerase), in nuclear DNA replication the correlation of their activity levels with the rate of nuclear DNA replication was determined and a comparison made of their catalytic properties. The experiments using either C3H2K cells, synchronized by serum starvation, or Ehrlich culture cells, arrested at the S phase by aphidicolin, showed DNA replicase to increase in cells in the S phase to at least six times that of the G0-phase cells but 7.3S polymerase to increase but slightly in this phase. This increase in DNA replicase activity most likely resulted from synthesis of a new enzyme, as shown by experiments using a specific monoclonal antibody, aphidicolin and cycloheximide. Not only with respect to the presence or absence of primase activity, but in other points as well the catalytic properties of these two forms were found to differ; DNA replicase preferred the activated calf thymus DNA with wide gaps of about 100 nucleotides long as a template-primer, while the optimal gap size for 7.3S polymerase was 40-50 nucleotides long. Size analysis of the products synthesized on M13 single-stranded circular DNA with a single 17-nucleotide primer by DNA replicase and 7.3S polymerase suggested the ability of DNA replicase to overcome a secondary structure formed in single-stranded DNA to be greater than that of 7.3S polymerase.     

Cyclic AMP regulates the expression and nuclear translocation of RFC40 in MCF7 cells

We have previously shown that the regulatory subunit of PKA, RIalpha, functions as a nuclear transport protein for the second subunit of the replication factor C complex, RFC40, and that this transport appears to be crucial for cell cycle progression from G1 to S phase. In this study, we found that N(6)-monobutyryl cAMP significantly up-regulates the expression of RFC40 mRNA by 1.8-fold and its endogenous protein by 2.3-fold with a subsequent increase in the RIalpha-RFC40 complex formation by 3.2-fold. Additionally, the nuclear to cytoplasmic ratio of RFC40 increased by 26% followed by a parallel increase in the percentage of S phase cells by 33%. However, there was reduction in the percentage of G1 cells by 16% and G2/M cells by 43% with a concurrent accumulation of cells in S phase. Interestingly, the higher percentage of S phase cells did not correlate with a parallel increase in DNA replication. Moreover, although cAMP did not affect the expression of the other RFC subunits, there was a significant decrease in the RFC40-37 complex formation by 81.3%, substantiating the decrease in DNA replication rate. Taken together, these findings suggest that cAMP functions as an upstream modulator that regulates the expression and nuclear translocation of RFC40.     

DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells

We have used C3H 10T1/2 cells to examine the regulation of topoisomerase activities during cell proliferation and the cell cycle. The specific activity of topoisomerase I was about 4-fold greater in proliferating (log phase) cells than in non-proliferating (confluent) cells. In synchronized cells, the bulk of the increased activity occurred during or just prior to S phase, depending upon the method of synchronization. A smaller increase in activity also occurred during G1 phase. The increase in activity during S phase was not altered by a hydroxyurea block at the G1/S phase boundary indicating that it is not directly coupled to DNA synthesis and is not the result of topoisomerase I gene dosage. The increase was inhibited by blocking cells at mid-G1 phase using isoleucine deprivation. Thus, the increase in activity during S phase is dependent on events occurring during mid- to late G1 phase. In contrast to the changes in topoisomerase I levels, the specific activity of topoisomerase II showed no detectable difference in proliferating vs non-proliferating cells. In addition, no detectable difference in topoisomerase II specific activity was seen in G1, S and M phases of the cell cycle. The differences in the activity profiles of the topoisomerases I and II during the cell cycle suggest that the two activities are regulated independently and may be required for different functions.     

Investigation on cell proliferation with a new antibody against thymidine kinase 1.

The cytosolic thymidine kinase 1 (TK1) is one of the enzymes involved in DNA replication. Based on biochemical studies, TK1 is activated at late G1 of cell cycle, and its activity correlates with the cell proliferation. We have developed a polyclonal anti-TK1 antibody against a synthetic peptide from the C-terminus of human TK1. Using this antibody, here we demonstrate the exclusive location of TK1 in the cytoplasm of cells. Cell cycle dependent TK1 expression was studied by simultaneous fluorescence staining for TK1 and bromodeoxyuridine, by using elutriated cells, and by quantitation of the amount TK1 in relation to the cellular DNA content. TK1, which was strongly expressed in the cells in S+G2 period, raised at late G1 and decreased during mitosis. The amount of TK1 increased three folds from late G1 to G2. TK1 positive cells were demonstrated in areas of proliferation activity of various normal and malignant tissues. The new anti-TK1 antibody works in archival specimens and is a specific marker of cell proliferation.     

Monoclonal antibody to calmodulin: development, characterization, and comparison with polyclonal anti-calmodulin antibodies

Specific anti-calmodulin rabbit polyclonal and murine monoclonal antibodies have been produced with a thyroglobulin-linked peptide corresponding to amino acids 128-148 of bovine brain calmodulin. The monoclonal antibody is IgG-1 with kappa light chains. Both sets of antibodies recognize native vertebrate calmodulin, with the polyclonal antibody exhibiting an approximately fourfold higher sensitivity than the monoclonal antibody in a radioimmunoassay. The affinity of both polyclonal and monoclonal antibodies is approximately 2.5-fold higher for Ca(2+)-free calmodulin than for Ca(2+)-calmodulin. Other selected members of the calmodulin family (S100, troponin, and parvalbumin) do not exhibit significant cross-reactivity with the monoclonal antibody. Troponin and S100 beta displace some 125I-calmodulin from the polyclonal antibody, but require at least 900-fold excess concentration. The monoclonal antibody recognizes intact vertebrate calmodulin in solution and also on solid-phase. In addition, plant calmodulin and some forms of post-translationally modified calmodulin (phosphorylated or glycated) bind the monoclonal antibody. The affinity of the monoclonal antibody is approximately 5 x 10(8) liters/mol determined by displacement of 125I-calmodulin. On dot blotting the sensitivity for vertebrate calmodulin is 50 pg. The epitope for the monoclonal antibody is in the carboxyl terminal region (residues 107-148) of calmodulin. This highly specific anti-calmodulin monoclonal antibody should be a useful reagent in elucidating the mechanism by which calmodulin regulates intracellular metabolism.     

Alteration of the Cloudman melanoma cell cycle by prostaglandins E1 and E2 determined by using a 5-bromo-2'-deoxyuridine method of DNA analysis

Prostaglandins (PGs) E1 and E2 stimulate tyrosinase activity and suppress the proliferation of Cloudman S91 melanoma cells by altering their progression through the cell cycle. Prostaglandin E1 and PGE2 have prolonged or residual effects on melanoma cells. Cells treated for 5 or 24 hours with 10 micrograms/ml PGE1 or cells treated for 8 or 24 hours with 10 micrograms/ml PGE2 demonstrated decreased proliferation and increased tyrosinase activity for 48 hours after removal of the PGs. The effects of PGs on the cell cycle were investigated by determining total DNA content in cells stained with propidium iodide (PI) and analyzed by a fluorescence activated cell sorter (FACS). Prostaglandin E1 blocked cells in G2 phase after 5 hours of treatment, corresponding to when inhibition of proliferation was first evident. Similarly, after 9 hours of treatment with PGE2, more cells were in late S, early G2 phase and less in G1 than their control counterparts. Also, melanoma cells were pulse-labeled with 5-bromo-2'-deoxyuridine (BrdUrd) prior to or at the end of PG treatment and then stained with a fluoresceinated monoclonal antibody to BrdUrd, and with PI. This allows one to observe how BrdUrd-labeled S-phase cells cycle with time. Both PGE1 and PGE2 inhibit proliferation by blocking cells in G2 phase of the cell cycle. The PG-induced block in G2 may be required by melanoma cells to synthesize mRNA and proteins that are essential for stimulation of tyrosinase activity. Ultrastructurally, only a subpopulation of the cells treated with PGE1 or PGE2 contained more mature melanosomes than control cells.     

The replication factory targeting sequence/PCNA-binding site is required in G(1) to control the phosphorylation status of DNA ligase I.

The recruitment of DNA ligase I to replication foci in S phase depends on a replication factory targeting sequence that also mediates the interaction with proliferating cell nuclear antigen (PCNA) in vitro. By exploiting a monoclonal antibody directed at a phospho-epitope, we demonstrate that Ser66 of DNA ligase I, which is part of a strong CKII consensus site, is phosphorylated in a cell cycle-dependent manner. After dephosphorylation in early G(1), the level of Ser66 phosphorylation is minimal in G(1), increases progressively in S and peaks in G(2)/M phase. The analysis of epitope-tagged DNA ligase I mutants demonstrates that dephosphorylation of Ser66 requires both the nuclear localization and the PCNA-binding site of the enzyme. Finally, we show that DNA ligase I and PCNA interact in vivo in G(1) and S phase but not in G(2)/M. We propose that dephosphorylation of Ser66 is part of a novel control mechanism to establish the pre-replicative form of DNA ligase I.     

Immunocytochemical localization of chick DNA polymerases alpha and beta +

An immunofluorescent method using specific antibodies was employed to detect DNA polymerases alpha and beta in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase alpha was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase beta, this enzyme was also shown to be present in nuclei. DNA polymerase alpha was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase beta were detected in proliferating and resting cells. Furthermore, DNA polymerase beta was detected in nuclei of most cells, while DNA polymerase alpha was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase alpha is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.     

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.     

Non-histone protein synthesis during G1 phase and its relation to DNA replication.

The kinetics of non-histone chromosomal protein (NHCP) synthesis were studied in Chinese hamster ovary (CHO) plateau phase cells stimulated to proliferate and were compared to NHCP synthesis kinetics in two populations of synchronous G1 traversing cells. In all cases, NHCP synthesis rates increase 3- to 5-fold as cells traversed G1 and attained maximum values one hour before semi-conservative DNA replication began. Similar to results in synchronous G1 cells, the molecular weight distributions of the NHCP fraction from stimulated plateau phase cells underwent only minor changes, measured by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, as these cells moved toward S phase. Yet, during this progression after plateau phase and in the transition from early G1 to late G1 in synchronous cells, the total NHCP fraction increased significantly (1.5-2-fold) in amount per cell. These data indicate that plateau phase cells are similar to early G1 cells both in terms of their amounts of non-histone per cell and in their subsequent NHCP synthesis kinetics as they move toward S phase. These results extend previous findings which suggested that NHCP synthesis was coupled to DNA replication and demonstrate that the increased NHCP synthesis and accumulation in chromatin may be a biochemical marker for G1 progression.     

Localization of ORC1 during the cell cycle in human leukemia cells

The interaction of the origin recognition complex (ORC) with replication origins is a critical parameter in eukaryotic replication initiation. In mammals the ORC remains bound except during mitosis, thus the localization of ORC complexes allows localization of origins. A monoclonal antibody that recognizes human ORC1 was used to localize ORC complexes in populations of human MOLT-4 cells separated by cell cycle position using centrifugal elutriation. ORC1 staining in cells in early G1 is diffuse and primarily peripheral. As the cells traverse G1, ORC1 accumulates and becomes more localized towards the center of the nucleus, however around the G1/S boundary the staining pattern changes and ORC1 appears peripheral. By mid to late S phase ORC1 immunofluorescence is again concentrated at the nuclear center. During anaphase, ORC1 staining is localized mainly in the pericentriolar regions. These findings suggest that concerted movements of origin DNA sequences in addition to the previously documented assembly and disassembly of protein complexes are an important aspect of replication initiation loci in eukaryotes.     

Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342,rabbit anti calmodulin antibody,FITC-labeled goat anti rabbit IgG and SR101(sulfo rhodamine 101)simultaneously to stain individual normal and transformed cells,the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus,calmodulin and total protein respectively,could be recognized in individualj cells without interference,The phase of the cell cycle was determined by DNA content(Hoechst 33342),We found that in transformed cells(NIH3T3) tsRSV-LA90,cultured at 33℃ and transformed C3H10T1/2 Cells),the ration of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV-LA90 cells,cultured at 39℃ and C3H10T1/2 cells)in every cell cycle phase,This ration increased obviously only from G1 to S phase in either normal or transformed cells.The results showed that calmodulinreally increased during the transformation,and its increase was specific.In the meantime when cells proceeded from G1 to S.the intraceollular calmodulin content also increased specifically.     

Chromatin structure restricts origin utilization when quiescent cells re-enter the cell cycle

Quiescent cells reside in G0 phase, which is characterized by the absence of cell growth and proliferation. These cells remain viable and re-enter the cell cycle when prompted by appropriate signals. Using a budding yeast model of cellular quiescence, we investigated the program that initiated DNA replication when these G0 cells resumed growth. Quiescent cells contained very low levels of replication initiation factors, and their entry into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase.     

Stockpiling of Cdc25 during a DNA replication checkpoint arrest in Schizosaccharomyces pombe.

The DNA replication checkpoint couples the onset of mitosis with the completion of S phase. It is clear that in the fission yeast Schizosaccharomyces pombe, operation of this checkpoint requires maintenance of the inhibitory tyrosyl phosphorylation of Cdc2. Cdc25 phosphatase induces mitosis by dephosphorylating tyrosine 15 of Cdc2. In this report, Cdc25 is shown to accumulate to a very high level in cells arrested in S. This shows that mechanisms which modulate the abundance of Cdc25 are unconnected to the DNA replication checkpoint. Using a Cdc2/cyclin B activation assay, we found that Cdc25 activity increased approximately 10-fold during transit through M phase. Cdc25 was activated by phosphorylations that were dependent on Cdc2 activity in vivo. Cdc25 activation was suppressed in cells arrested in G1 and S. However, Cdc25 was more highly modified and appeared to be somewhat more active in S than in G1. This finding might be connected to the fact that progression from G1 to S increases the likelihood that constitutive Cdc25 overproduction will cause inappropriate mitosis.