ABSTRACT
Cells experiencing DNA replication stress activate a response pathway that delays entry into mitosis and promotes DNA repair and completion of DNA replication. The protein kinases ScRad53 and SpCds1 (in baker's and fission yeast, respectively) are central to this pathway. We describe a conserved protein Mrc1, mediator of the replication checkpoint, required for activation of ScRad53 and SpCds1 during replication stress. mrc1 mutants are sensitive to hydroxyurea and have a checkpoint defect similar to rad53 and cds1 mutants. Mrc1 may be the replicative counterpart of Rad9 and Crb2, which are required for activating ScRad53 and Chk1 in response to DNA damage.
Subject(s)
DNA Replication , DNA, Fungal/biosynthesis , Fungal Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Cell Cycle Proteins/metabolism , Checkpoint Kinase 2 , Enzyme Activation , Fungal Proteins/genetics , Genes, Fungal , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Protein Kinases/metabolism , S Phase , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Schizosaccharomyces , Schizosaccharomyces pombe ProteinsABSTRACT
Epstein-Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP-dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co-immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.
Subject(s)
DNA Replication , DNA, Viral/biosynthesis , DNA-Binding Proteins/metabolism , Herpesvirus 4, Human/genetics , Replication Origin , Virus Latency , Virus Replication , Animals , B-Lymphocytes , Binding Sites , Epstein-Barr Virus Nuclear Antigens/metabolism , Herpesvirus 4, Human/physiology , Humans , Origin Recognition Complex , RatsSubject(s)
Cell Cycle/genetics , DNA Replication/drug effects , Hydroxyurea/pharmacology , Saccharomyces cerevisiae/genetics , Cell Cycle/drug effects , DNA Replication/genetics , DNA, Fungal/drug effects , DNA, Fungal/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effectsABSTRACT
In the past, eukaryotic cell-derived complexes of the Myc/Max/Mad network of transcriptional regulators have largely been refractory to DNA binding studies. We have developed electrophoretic mobility shift assay conditions to measure specific DNA binding of Myc/Max/Mad network complexes using a COS7 cell-based overexpression system. With the established protocol, we have measured on- and off-rates of c-Myc/Max, Max/Max, and Mad1/Max complexes and determined relative affinities. All three complexes appeared to bind with comparable affinity to a Myc E-box sequence. Furthermore, our data derived from competition experiments suggested that the Mad3/Max and Mad4/Max complexes also possess comparable DNA binding affinities. The conditions established for COS7 cell-overexpressed proteins were then used to identify c-Myc/Max, Max/Max, and Mnt/Max complexes in HL-60 cells. However, no Mad1/Max could be detected, despite the induction of Mad1 expression during differentiation. Whereas the DNA binding activity of c-Myc/Max complexes was down-regulated, Max/Max binding increased, and Mnt/Max binding remained unchanged. In addition, we have also tested for upstream stimulatory factor (USF) binding and observed that, in agreement with published data, USF comprises a major Myc E-box-binding factor that is more abundant than any of the Myc/Max/Mad network complexes. Similar to the Mnt/Max complex, the binding activity of USF remained constant during HL-60 differentiation. Our findings establish conditions for the analysis of DNA binding of Myc/Max/Mad complexes and indicate posttranslational regulation of the Max/Max complex.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Repressor Proteins , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , COS Cells , HL-60 Cells , Humans , Protein BindingABSTRACT
The Cdc7p protein kinase plays an essential, but undefined, role promoting S phase in the budding yeast, Saccharomyces cerevisiae. Previous experiments have shown that the essential function of Cdc7 is executed near the G1-S boundary; after Start but before the elongation phase of DNA replication. Origins of DNA replication fire throughout S phase in budding yeast. Therefore, the G1-S transition is a cell-cycle event that precedes, and is distinct from, the activation of individual origins. Consequently, we have asked whether Cdc7 is only required for S-phase entry or if it plays a role during S phase in origin firing. In this article, we show that partial loss of Cdc7 function results in slow progression through S phase rather than slow entry into S phase and that Cdc7 is still required for the timely completion of S phase after a block to elongation with hydroxyurea. This is because Cdc7 is still required for the activation of late-firing origins after the hydroxyurea block. These experiments show that, rather than acting as a global regulator of the G1-S transition, Cdc7 appears to play a more direct role in the firing of replication origins during S phase.
Subject(s)
Cell Cycle Proteins/metabolism , DNA Replication , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Replication Origin , S Phase/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Cell Cycle Proteins/genetics , DNA Footprinting , DNA Replication/drug effects , Flow Cytometry , G1 Phase , Hydroxyurea/pharmacology , Mutation , Nucleic Acid Synthesis Inhibitors/pharmacology , Plasmids/genetics , Protein Kinases/geneticsABSTRACT
c-Myc is an essential component of the regulatory mechanisms controlling cell growth. Max is the obligatory partner of c-Myc for all its biological functions analysed to date. Recently two Max interacting proteins, Mad and Mxi1, have been identified. It has been suggested that these two proteins modulate c-Myc function, in the simplest model by competing with c-Myc for the interaction with Max. We have analysed different aspects of Mad function in comparison to Max. Native Mad/Max heterodimers bound specifically to a c-Myc/Max consensus DNA binding site. Furthermore Mad inhibited efficiently c-Myc, mutant p53, adenovirus E1a, or human papilloma virus type 16 transformation of rat embryo cells in cooperation with activated Ha-Ras. Myc transformed clones showed an increased cell cycle time and a reduced immortalization frequency after cotransfection with either mad or max. In contrast to Mad, Max did not inhibit E1a/Ha-Ras cotransformation but repressed c-Myc/Ha-Ras transformation efficiently. Mad delta N, an N-terminal deletion mutant of Mad, was as efficient in repressing c-Myc/Ha-Ras cotransformation as full length Mad but showed little inhibitory activity when assayed on E1a/Ha-Ras. Unlike wt Mad, Mad delta N had little effect on cell growth. Our data suggest that Mad affects cell growth at least in part by a c-Myc independent mechanism.
Subject(s)
Cell Transformation, Neoplastic , DNA-Binding Proteins/physiology , Oncogene Proteins, Viral/physiology , Proto-Oncogene Proteins c-myc/physiology , Proto-Oncogene Proteins p21(ras)/physiology , Repressor Proteins , Transcription Factors , Animals , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Cell Cycle , Cell Line , Chlorocebus aethiops , Gene Expression , Genes, myc , Genes, ras , Macromolecular Substances , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/chemistry , RNA, Messenger/genetics , Structure-Activity Relationship , TransfectionABSTRACT
A number of transcription factors have been shown to be phosphorylated by casein kinase II (CKII). We have identified CKII phosphorylation sites in c-Myc, Max, and c-Myb which are phosphorylated in the cell. Whereas little evidence to any functional significance of the CKII sites in c-Myc has been obtained, phosphorylation of its heterodimeric partner Max alters DNA binding properties. CKII phosphorylation of Ser-2 and -11 in Max resulted in enhanced DNA binding kinetics of both Max/Max homo- and Myc/Max heterodimers without altering steady state binding. Replacing these serine by alanine residues and comparing the wild type with the mutant Max proteins in transactivation assays did not reveal any significant differences. For c-Myb mutational analysis of the CKII phosphorylation sites showed altered steady state DNA binding. Replacing Ser-11/12 by alanine residues resulted in increased DNA binding compared to wt c-Myb or Myb Asp-11/12 as demonstrated by up to 10-fold differences in the dissociation constants. In transactivation assays, the Ala mutant showed consistently an increased activity both on a synthetic and on the mim-1 promoter. A potential CKII phosphorylation site in c-Fos was not phosphorylated in vitro. Analysis with peptides demonstrated that a proline residue at position +1 relative to the acceptor serine was inhibitory.
Subject(s)
DNA-Binding Proteins/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins c-myc/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Casein Kinase II , Cell Line , Cell Line, Transformed , Chlorocebus aethiops , Genes, Reporter , Helix-Loop-Helix Motifs , Kidney , Molecular Sequence Data , Mutagenesis, Site-Directed , PC12 Cells , Phosphorylation , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-myb , Rabbits , Rats , Recombinant Fusion Proteins/metabolism , Transcriptional Activation , TransfectionABSTRACT
Myc proteins have been implicated in the regulation of cell growth and differentiation. The identification of Max, a basic region/helix-loop-helix/leucine zipper protein, as a partner for Myc has provided insights into Myc's molecular function as a transcription factor. Recent evidence indicates that the relative abundance of Myc and Max is important to determine the level of specific gene transcription. In this report we have identified two major in vivo phosphorylation sites in Max (Ser-2 and -11) which can be modified in vitro by casein kinase II (CKII). Phosphorylation of these sites modulates DNA-binding by increasing both the on- and off-rates of Max homo- as well as Myc/Max heterodimers. In addition, our data indicate that the steady state binding of the shorter version of Max (p21) to DNA was similar yet its rate of dissociation faster than that of longer version of Max (p22). These data argue that different Max complexes have different kinetic properties and that these can be modified by CKII phosphorylation. We propose this as an important biological mechanism by which different dimeric complexes can exchange with varying efficiencies on DNA, thereby responding to changes in cell growth conditions.
Subject(s)
DNA, Neoplasm/metabolism , DNA-Binding Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors , Amino Acid Sequence , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Basic-Leucine Zipper Transcription Factors , Burkitt Lymphoma/genetics , Burkitt Lymphoma/pathology , Casein Kinase II , Cell Division/physiology , DNA, Neoplasm/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Peptide Mapping , Phosphorylation , Precipitin Tests , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/physiology , Serine/analysis , Serine/physiology , Transcription, Genetic/genetics , Tumor Cells, CulturedABSTRACT
In Neisseria meningitidis, translocation of capsular polysaccharides to the cell surface is mediated by a transport system that fits the characteristics of ABC (ATP-binding cassette) transporters. One protein of this transport system, termed CtrA, is located in the outer membrane. By use of a CtrA-specific monoclonal antibody, we could demonstrate that CtrA occurs exclusively in N. meningitidis and not in other pathogenic or nonpathogenic Neisseria species. Nucleotide sequence comparison of the ctrA gene from different meningococcal serogroups indicated that CtrA is strongly conserved in all meningococcal serogroups, independent of the chemical composition of the capsular polysaccharide. Secondary structure analysis revealed that CtrA is anchored in the outer membrane by eight membrane-spanning amphipathic beta strands, a structure of proteins that function as porins.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Neisseria meningitidis/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/immunology , Base Sequence , Epitopes/analysis , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neisseria meningitidis/immunology , Protein ConformationABSTRACT
Capsular polysaccharides of Gram-negative bacteria contribute to a large extent to the pathogenicity of these organisms. We show here that the molecular organization of the capsule gene loci in different serogroups of Neisseria meningitidis is similar to that of Haemophilus influenzae and Escherichia coli. A common molecular origin of the mechanisms of encapsulation is indicated by strong homology of the genes involved in transport of the capsular polysaccharides to the cell surface in all these organisms. The proteins involved in capsular polysaccharide transport fit the characteristics of ABC (ATP-binding cassette) transporters. Furthermore, by sequence comparison of the sialytransferases of N. meningitidis B and E. coli K1, the capsule of which is composed of alpha 2,8-linked polyneuraminic acid, a significant degree of homology was observed, indicating that the capsular polysaccharide type itself has the same evolutionary origin in these two pathogens.