A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication

DNA replication in eukaryotic cells initiates from many replication origins which fire throughout the S phase of the cell cycle in a predictable pattern: some origins fire early, others late. Little is known about how the initiation of DNA replication and the elongation of newly synthesized DNA strands are coordinated during S phase. Here we show that, in budding yeast, hydroxyurea, which blocks the progression of replication forks from early-firing origins, also inhibits the firing of late origins. These late origins are maintained in the initiation-competent prereplicative state for extended periods. The block to late origin firing is an active process and is defective in yeast with mutations in the rad53 and mec1 checkpoint genes, indicating that regulation of late origin firing may also be an important component of the 'intra-S-phase' checkpoint and may aid cell survival under adverse conditions.     

Schizosaccharomyces pombe Mcm3p, an essential nuclear protein, associates tightly with Nda4p (Mcm5p)

MCM proteins are required for the proper regulation of DNA replication. There are six MCM proteins in all eukaryotes which interact to form a large complex. We report the cloning of fission yeast mcm3 +. mcm3 + is essential and spores carrying a Delta mcm3 disruption arrest with an apparently replicated DNA content. The protein is found constitutively in the nucleus and levels remain constant throughout the cell cycle. Mcm3p binds particularly tightly to Nda4p (Mcm5p), but is loosely associated with the other Schizosaccharomyces pombe MCM proteins. Thus, Mcm3p is a peripheral MCM subunit.     

Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication

We report the isolation and characterization of CDC45, which encodes a polypeptide of 650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. Like mutants in several MCM genes, alleles of CDC45 also show a severe minichromosome maintenance defect. Together, these observations imply that Cdc45p performs a role in the control of initiation events at chromosomal replication origins. We investigated this possibility further and present evidence demonstrating that Cdc45p is assembled into complexes with one MCM family member, Cdc46p/Mcm5p. These observations point to a role for Cdc45p in controlling the early steps of chromosomal DNA replication in conjunction with MCM polypeptide complexes. Unlike the MCMs, however, the subcellular localization of Cdc45p does not vary with the cell cycle, making it likely that Cdc45p interacts with MCMs only during the nuclear phase of MCM localization in G1.     

Regulation of Myc-dependent apoptosis by p53, c-Jun N-terminal kinases/stress-activated protein kinases, and Mdm-2

Deregulated overexpression of c-Myc (Myc) confers susceptibility to apoptosis in several cell types, but the molecular regulation of these processes has not been well established. Here we have characterized several molecular changes that may modulate Myc-dependent apoptosis. Ectopic overexpression of Myc in both Rat1 fibroblasts and human osteosarcoma cells causes a dramatic increase of cellular p53 mRNA and protein, and this induction of p53 correlates with apoptosis triggered by withdrawal of serum. Stable transfection of a wild-type human p53 gene into Myc-transformed cells further potentiates apoptosis. Anticancer agents vinblastine and nocodazole also induce apoptosis in Myc-transformed Rat1 fibroblasts but are cytostatic to the same cells without Myc overexpression. We demonstrate that induction of Myc-dependent apoptosis in these cells is specifically associated with an activation of p46 c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activity, whereas this JNK/SAPK activation is absent in stress-treated cells without Myc overexpression. Moreover, overexpression of the Mdm-2 gene in Rat1-myc cells significantly inhibits apoptosis induced by low serum but has little effect on apoptosis triggered by chemotherapeutic drugs. Interestingly, differential inhibition by Mdm-2 paralleled differential activation of p46 JNK/SAPK. Thus, our data support a functional involvement of p53 in Myc-dependent apoptosis and implicate potential regulatory roles for JNK/SAPK and Mdm-2 pathways in the regulation of apoptosis in Myc-transformed tumor cells.     

Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actin-crosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5-10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin.     

p53 and RPA are sequestered in viral replication centers in the nuclei of cells infected with human cytomegalovirus

Previously, we reported that human cytomegalovirus (HCMV) infection of fibroblasts markedly affects p53 and other regulatory proteins and inhibits transit through the cell cycle (F. M. Jault, J.-M. Jault, F. Ruchti, E. A. Fortunato, C. Clark, J. Corbeil, D. D. Richman, and D. H. Spector, J. Virol. 69:6697-6704, 1995). Although the p53 steady-state levels are elevated throughout the infection, evidence suggests that the ability of p53 to transactivate some of its downstream targets is compromised. To elucidate the mechanisms governing the accumulation of p53, we examined the synthesis, stability, and localization of the protein in HCMV-infected fibroblasts. Synthesis of p53 was not increased in the infected cells during the first 24 h postinfection. In fact, pulse-chase experiments revealed that synthesis of p53 in infected fibroblasts was lower than in mock-infected cells. However, after an initial decay, the p53 was stabilized. In addition, beginning at approximately 30 h postinfection, p53 was localized to discrete foci within the nuclei of infected cells. The morphology of these foci suggested that they were replication centers. We confirmed that these are sites of DNA replication by demonstrating both incorporation of bromodeoxyuridine and localization of UL44 (the viral polymerase processivity factor) into these centers. The single-stranded DNA binding protein RPA was also sequestered. In contrast, Rb and HCMV IE1 72 remained distributed throughout the infected cell nuclei, indicating specific targeting of certain proteins. Taken together, our results provide two alternative mechanisms to account for the increased steady-state levels of p53 observed in HCMV-infected fibroblasts.     

Intracellular localization of p53 tumor suppressor protein in gamma-irradiated cells is cell cycle regulated and determined by the nucleus

DNA damage leads to the stabilization of p53 protein and its translocation to the nucleus, resulting in activation or suppression of p53-responsive genes. However, a significant proportion of cell nuclei remain negative for p53 and p53-inducible cyclin-dependent kinase inhibitor p21waf1 after a single dose of gamma-irradiation. Quantitation of DNA content in p53-positive and -negative nuclei 4-6 h after 10 Gy of gamma-irradiation of human breast carcinoma MCF7 cells, fibrosarcoma HT1080 cells, and diploid skin fibroblasts showed that p53 and p21waf1 nuclear accumulation occurs predominantly in the G1 phase and at the beginning of the S phase of the cell cycle. The majority of the nuclei in late S phase and in G2-M phase remained p53- and p21waf1-negative. This suggests that there is a cell cycle window during which p53 can accumulate in the nucleus and activate expression of p21waf1. To determine whether cell cycle-dependent distribution of p53 is caused by cytoplasmic modifications of p53 protein or by properties of the nucleus, p53 localization was analyzed in multinucleated cells obtained by polyethylene glycol-mediated cell fusion. Dramatic differences in p53 accumulation were found among the nuclei in individual multinucleated cells. Distribution of p53-positive and -negative nuclei among the phases of the cell cycle was similar to that observed in a regular cell population. These results suggest that the observed differences in p53 accumulation in the nuclei of irradiated cells are determined by cell cycle-dependent nuclear functions. In contrast to p53, p21waf1 was equally distributed among the nuclei of multinucleated cells regardless of the stage of the cell cycle, indicating that the observed phenomenon is specific for p53.     

Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.     

Effect on DNA topology by DnaA protein, the initiation factor of chromosomal DNA replication in Escherichia coli

We examined effects on supercoiled DNA topology of DnaA protein, the initiator protein of chromosomal DNA replication in Escherichia coli. The activity was identified in an analysis of plasmid DNA incubated with DnaA protein and DNA topoisomerase I. In Superose 12 gel filtration chromatography, the activity coeluted with DnaA protein. Incubation of DnaA protein with DNA at temperatures over 24 degrees C was required for this activity, which was observed with either oriC plasmid or the replicative form I of phi X174 with no DnaA box. As binding of ATP or ADP to DnaA protein prevented the activity of DnaA protein on DNA topology, binding of the adenine nucleotide may regulate the activity.     

DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.     

CD4 T cell tolerance to human C-reactive protein, an inducible serum protein, is mediated by medullary thymic epithelium

In this study we examined the production of gelatinases A and B (MMP-2 and MMP-9), and their natural inhibitors TIMP-1 and TIMP-2 in cell lines derived from different histologic types of lung cancer. Gelatinolytic activity was measured by zymography and radiolabeled gelatin degradation. Immunocytochemistry and Western blot analysis were performed to corroborate the presence of immunoreactive MMP-2, MMP-9, TIMP-1 and TIMP-2 proteins. The highest gelatinolytic activity was identified in the cell extracts from a small-cell carcinoma cell line. MMP-9 was observed in all samples as a proenzyme, while MMP-2 was present as zymogen in the squamous-cell and in the small-cell carcinomas, and in its active form in one squamous-cell carcinoma cell line. TIMPs were also present in the neoplastic lung cell lines. TIMP-1 was observed in the media of all cells as a 21-kD band, and as TIMP-1 polymers with the exception of the small-cell carcinoma samples. TIMP-2 was found as higher-order molecular immunoreactive complexes that may correspond to proMMP-2/TIMP-2 complexes. These results demonstrate that lung neoplastic cells produce both MMP-2 and MMP-9 and their inhibitors, with the small-cell carcinoma cell extracts showing the highest enzymatic activity. This gelatinolytic activity fits well with the clinical metastatic behavior of this type of lung cancer.     

E1B 19,000-molecular-weight protein interacts with and inhibits CED-4-dependent, FLICE-mediated apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-xL) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-xL have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Association of protein kinases ERK1 and ERK2 with p75 nerve growth factor receptors

Extracellular signal-regulated protein kinases (ERKs) constitute a family of protein serine-threonine kinases implicated in a variety of cell-signaling pathways. In cultured rat pheochromocytoma PC12 cells, ERK1 and ERK2 are activated by nerve growth factor (NGF), which also induces rapid association between ERK1 and the high affinity gp140prototrk tyrosine kinase NGF receptor. In the present work, we investigated the possible association between ERKs and the low affinity NGF receptor, p75. Extracts of PC12 cells (before and after NGF treatment) were subjected to immunoprecipitation with anti-p75 antibodies or antiserum; the immune complexes were then assessed for the presence of ERK proteins and tyrosine phosphorylation or for ERK activity using a specific substrate peptide. ERK1 and, to a lesser extent, ERK2 were found to be constitutively associated with p75. NGF did not modulate the total amount of ERK proteins coimmunoprecipitated with p75 but did markedly stimulate the level of p75-associated ERK catalytic activity. NGF treatment also enhanced the tyrosine phosphorylation of a p75-associated species that co-migrates with ERK1 in Western blots. Finally, K-252a, a compound that specifically inhibits activation by NGF of gp140prototrk, abolished the latter effect. These findings indicate that NGF, via activation of gp140prototrk, leads to association of enzymatically active ERKs with p75 and raise the possibility that this interaction may play a role in the NGF mechanism of action.