Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.22 (cdc2)
 8,319 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used the antisense strategy to study the role of certain genes in cell cycle progression. In particular, we used antisense oligodeoxynucleotides to study: (1) the role of the IGF-1 receptor in the control of cell proliferation; and (2) the sequence of gene expression during the cell cycle. Our results can be summarized as follows: (1) the activation of the IGF-1 receptor by its ligand, IGF-1, is an obligatory step in the proliferation of fibroblasts and hemopoietic cells; and (2) the expression of DNA synthesis genes, such as PCNA, DNA polymerase alpha, and cdc2, is dependent on the expression of previous genes. A tentative temporal order is: c-myc > c-myb > IGF-1 receptor > DNA synthesis genes.
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PMID:Inhibition of cell cycle progression by antisense oligodeoxynucleotides. 134 Jan 57

Saccharomyces cerevisiae cdc2 mutants arrest in the S-phase of the cell cycle when grown at the non-permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3'-5' exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.
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PMID:Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. 267 May 63

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.
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PMID:Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells. 762 1

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.
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PMID:Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes. 762 16

Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.
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PMID:Cyclin/cdk2 complexes in the nucleus of HeLa cells. 794 2

We have investigated the capacity of a murine cell line with a temperature-sensitive (ts) mutation in the DNA polymerase alpha (Pola) locus and a series of ts non-Pola mutant cell lines from separate complementation groups to stimulate DNA synthesis, in senescent fibroblast nuclei in heterokaryons. In the Pola mutant x senescent heterodikaryons, both human and murine nuclei display significantly diminished levels of DNA synthesis at the restrictive temperature (39.5 degrees C) as determined by [3H]thymidine labeling in autoradiographs. In contrast, all of the non-Pola mutants, as well as the parental (wild type) murine cells, induced similar levels of DNA synthesis in both parental nuclei at the nonpermissive and permissive temperatures. Similarly, young human fibroblasts are also able to initiate DNA synthesis in heterokaryons with the ts Pola mutant at the two temperatures. In order to determine if complementation of the non-Pola mutants requires induction of serum responsive factors in the senescent cells, fusion studies of similar design were conducted with young and old human fibroblasts incubated in low serum (0.2%) for 48 hr prior to and after cell fusion. Again, a diminished level of DNA synthesis was observed at 39.5 degrees C in the Pola mutant x senescent cell heterokaryons. In these low-serum studies, both parental nuclei in the Pola x young cell heterokaryons and the human nuclei in heterokaryons with one of the non-Pola mutants (FT107) also displayed diminished levels of DNA synthetic activity. All of the other mutants are able to support similar levels of synthetic activity at both temperatures in the presence of reduced serum. The nature of the mutation in three of the non-Pola lines has not been determined but, like the Pola mutant cells, are inhibited in the G1 phase of the cell cycle when incubated at the nonpermissive temperature (39.5 degrees C). The fourth non-Pola mutant line is known to have at least one ts mutation in the cdc2 gene and is inhibited in the G2 phase when exposed to 39.5 degrees C. These results suggest that there may be a functional deficiency of pol alpha in senescent human fibroblasts, and this replication factor may be one of the rate-limiting factors involved in loss of the capacity to initiate DNA synthesis in senescent cells.
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PMID:Murine temperature-sensitive DNA polymerase alpha mutant displays a diminished capacity to stimulate DNA synthesis in senescent human fibroblast nuclei in heterokaryons at the nonpermissive condition. 810 64

We studied the phosphorylation of fission yeast p170 (the catalytic subunit of DNA polymerase alpha) and its relationship to the cell cycle. In exponentially growing cells, p170 was phosphorylated at serine residues. Its phosphorylation level did not quantitatively change when cell strains carrying conditional cell division cycle (cdc) mutations arrested at different stages of the cell cycle, under restrictive growth conditions. Especially, phosphorylation did not significantly vary when cells carrying the temperature-sensitive cdc2-33 mutation were shifted to the restrictive temperature, which indicates a minor role, if any, of p34cdc2 in this process. Also, the extent of p170 phosphorylation did not remarkably change during mitosis, a situation which differs from that reported for human DNA polymerase alpha. We used immunofluorescence microscopy and cell fractionation to study the intracellular distribution of p170. We here provide evidence that the protein remains tenaciously associated with nuclear structures throughout the cell cycle and is not redistributed into the cytoplasm at mitosis, as it is in human cells. A possible correlation between phosphorylation, nuclear binding, and mitotic behavior of DNA polymerase alpha catalytic subunits in eukaryotes is therefore conceivable.
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PMID:In vivo phosphorylation, mitotic behavior, and nuclear binding of the catalytic subunit of DNA polymerase alpha in fission yeast. 831 72

DNA polymerases alpha and delta are essential enzymes believed to play critical roles in initiation and replication of chromosome DNA. In this study, we show that the genes for Schizosaccharomyces pombe (S.pombe) DNA polymerase alpha and delta (pol alpha+ and pol delta+) are essential for cell viability. Disruption of either the pol alpha+ or pol delta+ gene results in distinct terminal phenotypes. The S.pombe pol delta+ gene is able to complement the thermosensitive cdc2-2 allele of Saccharomyces cerevisiae (S.cerevisiae) at the restrictive temperature. By random mutagenesis in vitro, we generated three pol delta conditional lethal alleles. We replaced the wild type chromosomal copy of pol delta+ gene with the mutagenized sequence and characterized the thermosensitive alleles in vivo. All three thermosensitive mutants exhibit a typical cell division cycle (cdc) terminal phenotype similar to that of the disrupted pol delta+ gene. Flow cytometric analysis showed that at the nonpermissive temperature all three mutants were arrested in S phase of the cell cycle. The three S.pombe conditional pol delta alleles were recovered and sequenced. The mutations causing the thermosensitive phenotype are missense mutations. The altered amino acid residues are uniquely conserved among the known polymerase delta sequences.
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PMID:Fission yeast with DNA polymerase delta temperature-sensitive alleles exhibits cell division cycle phenotype. 836

The DNA polymerase alpha-primase complex purified from mouse FM3A cells is composed of four polypeptides with molecular masses of 180, 68, 54, and 46 kDa. The largest subunit has DNA polymerase activity, the two smallest subunits have DNA primase activity, and the function of the 68-kDa subunit is unknown. We have isolated the cDNAs of the four subunits by low stringency hybridization and reverse transcription polymerase chain reaction and determined their nucleotide sequences. The predicted amino acid sequence of the 180-kDa subunit shows 88, 38, 34, and 32% identity to those of the catalytic subunits of human, Drosophila melanogaster, Schizosaccharomyces pombe, and Saccharomyces cerevisiae DNA polymerase alpha, respectively, and contains seven regions whose orders and sequences are highly conserved among viral and other eukaryotic DNA polymerases. The deduced amino acid sequence of the 68-kDa subunit shows 25% identity to that of the 73-kDa subunit of D. melanogaster DNA polymerase alpha-primase, shows no significant sequence similarity to any other protein in the data bases, but contains a potential phosphorylation site(s) for cdc2 kinase. The amino acid sequence of the 54-kDa subunit shows 32% identity to that of the large subunit of S. cerevisiae DNA primase. During activation of quiescent Swiss mouse 3T3 cells to proliferate, the levels of mRNA of the four subunits of the DNA polymerase alpha-primase complex increased before DNA synthesis. In growing mouse FM3A cells, the transcripts of the four subunits are present throughout the cell cycle and increase slightly prior to the S phase.
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PMID:Molecular cloning of the cDNAs for the four subunits of mouse DNA polymerase alpha-primase complex and their gene expression during cell proliferation and the cell cycle. 846 24

The amino terminus of the E2F1 transcription factor is a protein-protein interaction domain since it associates with cyclin A/cdk2. Here, the two-hybrid yeast screen was used to clone genes whose products associate with the amino terminus of E2F1. The amino-terminal 121 amino acids of E2F1 were fused to the Lex A binding domain while a partial length cDNA library from the embryo of a 12 day old mouse was fused to the VP16 activation domain. Following coexpression of these fusions in yeast, two novel genes were cloned that code for proteins that associate with E2F1. In an in vitro assay, these E2F1 Binding Proteins (EBP1 and EBP2) associate with residues 1-121 of E2F1 or with the full-length protein; however, they do not associate with its carboxy terminus (residues 88-437). When EBP1 or EBP2 were expressed in COS cells along with E2F1 and the target promoter DNA polymerase alpha, repression of transcription was observed. However, no repression of DNA polymerase alpha was seen if the cells expressed a nonassociating mutant E2F1 (residues 88-437), along with EBP1 or EBP2. Finally, expression of the EBP2 gene is up-regulated in growing NIH3T3 fibroblasts, relative to serum-starved cells. However, this up-regulation of EBP2 expression is not seen in fibroblasts constitutively expressing E2F1.
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PMID:Isolation of two novel cDNAs whose products associate with the amino terminus of the E2F1 transcription factor. 882 66


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