scholarly journals Cdt1 Phosphorylation by Cyclin A-dependent Kinases Negatively Regulates Its Function without Affecting Geminin Binding

2004 ◽  
Vol 279 (19) ◽  
pp. 19691-19697 ◽  
Author(s):  
Nozomi Sugimoto ◽  
Yasutoshi Tatsumi ◽  
Tatsuya Tsurumi ◽  
Akio Matsukage ◽  
Tohru Kiyono ◽  
...  
Keyword(s):  
Origin Recognition Complex ◽  
Cyclin A ◽  
Binding Activity ◽  
Binding Motif ◽  
Minichromosome Maintenance ◽  
Dna Binding Activity ◽  
Cdk Phosphorylation ◽  
F Box Protein ◽  
Origin Recognition

The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G1phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast,in vitroDNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G2/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.

scholarly journals DrosophilaHeterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

2001 ◽  
Vol 12 (6) ◽  
pp. 1671-1685 ◽  
Author(s):  
Mohammed Momin Shareef ◽  
Chadwick King ◽  
Mona Damaj ◽  
RamaKrishna Badagu ◽  
Da Wei Huang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Origin Recognition Complex ◽  
Polytene Chromosomes ◽  
Binding Activity ◽  
Hmg Proteins ◽  
Mitotic Chromosomes ◽  
Origin Firing ◽  
Dna Binding Activity ◽  
Origin Recognition

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes inSaccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophilaembryo is associated with a multiprotein complex containingDrosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

scholarly journals Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro

2000 ◽  
Vol 14 (13) ◽  
pp. 1631-1641 ◽  
Author(s):  
Tohru Mizushima ◽  
Naoko Takahashi ◽  
Bruce Stillman
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Origin Recognition Complex ◽  
Binding Activity ◽  
Chromosomal Dna ◽  
Dna Binding Activity ◽  
Dna Binding Specificity ◽  
Dna Replication Origins ◽  
Origin Recognition

An interaction between the origin recognition complex (ORC) and Cdc6p is the first and a key step in the initiation of chromosomal DNA replication. We describe the assembly of an origin-dependent complex containing ORC and Cdc6p from Saccharomyces cerevisiae. Cdc6p increases the DNA binding specificity of ORC by inhibiting non-specific DNA binding of ORC. Cdc6p induces a concomitant change in the conformation of ORC and mutations in the Cdc6p Walker A and Walker B motifs, or ATP-γ-S inhibited these activities of Cdc6p. These data suggest that Cdc6p modifies ORC function at DNA replication origins. On the basis of these results in yeast, we propose that Cdc6p may be an essential determinant of origin specificity in metazoan species.

scholarly journals Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation.

1994 ◽  
Vol 14 (12) ◽  
pp. 8420-8431 ◽  
Author(s):  
M Xu ◽  
K A Sheppard ◽  
C Y Peng ◽  
A S Yee ◽  
H Piwnica-Worms
Keyword(s):  
Dna Binding ◽  
Active Form ◽  
Cyclin A ◽  
Binding Activity ◽  
Stable Complex ◽  
Binding Studies ◽  
Dna Binding Activity ◽  
Phosphopeptide Mapping

E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G1-to-S phase transition in higher eukaryotic cells. E2F-1 forms a heterodimer with DP-1 and binds to several cell cycle regulatory proteins, including the retinoblastoma family (RB, p107, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphorylation and its interaction with cyclin A/CDK2 complexes both in vivo and in vitro. In vitro, E2F-1 formed a stable complex with cyclin A/CDK2 but not with either subunit alone. DP-1 did not interact with cyclin A, CDK2, or the cyclin A/CDK2 complex. While the complex of cyclin A/CDK2 was required for stable complex formation with E2F-1, the kinase-active form of CDK2 was not required. However, E2F-1 was phosphorylated by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa cells. Two-dimensional tryptic phosphopeptide mapping studies demonstrated an overlap in the phosphopeptides derived from E2F-1 labeled in vitro and in vivo, indicating that cyclin A/CDK2 may be responsible for the majority of E2F-1 phosphorylation in vivo. Furthermore, an active DNA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo demonstrated that the cyclin A/CDK2-binding region resided within the N-terminal 124 amino acids of E2F-1. Because the stable association of E2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- or RB-binding domain and because the interactions could be reconstituted from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that the DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation by cyclin A/CDK2.

scholarly journals Identification of the varR Gene as a Transcriptional Regulator of Virginiamycin S Resistance inStreptomyces virginiae

2001 ◽  
Vol 183 (6) ◽  
pp. 2025-2031 ◽  
Author(s):  
Wises Namwat ◽  
Chang-Kwon Lee ◽  
Hiroshi Kinoshita ◽  
Yasuhiro Yamada ◽  
Takuya Nihira
Keyword(s):  
Dna Binding ◽  
Promoter Region ◽  
Northern Blot Analysis ◽  
Binding Activity ◽  
Binding Motif ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Dna Binding Activity ◽  
Streptomyces Virginiae

ABSTRACT A gene designated varR (for virginiaeantibiotic resistance regulator) was identified in Streptomyces virginiae 89 bp downstream of a varS gene encoding a virginiamycin S (VS)-specific transporter. The deduced varRproduct showed high homology to repressors of the TetR family with a conserved helix-turn-helix DNA binding motif. Purified recombinant VarR protein was present as a dimer in vitro and showed clear DNA binding activity toward the varS promoter region. This binding was abolished by the presence of VS, suggesting that VarR regulates transcription of varS in a VS-dependent manner. Northern blot analysis revealed that varR was cotranscribed with upstream varS as a 2.4-kb transcript and that VS acted as an inducer of bicistronic transcription. Deletion analysis of thevarS promoter region clarified two adjacent VarR binding sites in the varS promoter.

scholarly journals Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

1998 ◽  
Vol 142 (2) ◽  
pp. 307-318 ◽  
Author(s):  
Da Wei Huang ◽  
Laura Fanti ◽  
Daniel T.S. Pak ◽  
Michael R. Botchan ◽  
Sergio Pimpinelli ◽  
...  
Keyword(s):  
Origin Recognition Complex ◽  
Binding Activity ◽  
Heterochromatin Protein 1 ◽  
Dna Binding Activity ◽  
Nucleation Sites ◽  
Repressive Effect ◽  
Drosophila Heterochromatin ◽  
Drosophila Heterochromatin Protein ◽  
Origin Recognition

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.

scholarly journals Quantitative Proteomic Identification of MAZ as a Transcriptional Regulator of Muscle-Specific Genes in Skeletal and Cardiac Myocytes

2008 ◽  
Vol 28 (20) ◽  
pp. 6521-6535 ◽  
Author(s):  
Charis L. Himeda ◽  
Jeffrey A. Ranish ◽  
Stephen D. Hauschka
Keyword(s):  
Cardiac Myocytes ◽  
Binding Activity ◽  
Binding Motif ◽  
Gene Promoters ◽  
Muscle Creatine Kinase ◽  
Conserved Sequence ◽  
Dna Binding Activity ◽  
Myocyte Differentiation ◽  
Muscle Gene

ABSTRACT We identified a conserved sequence within the Muscle creatine kinase (MCK) promoter that is critical for high-level activity in skeletal and cardiac myocytes (MCK Promoter Element X [MPEX]). After selectively enriching for MPEX-binding factor(s) (MPEX-BFs), ICAT-based quantitative proteomics was used to identify MPEX-BF candidates, one of which was MAZ (Myc-associated zinc finger protein). MAZ transactivates the MCK promoter and binds the MPEX site in vitro, and chromatin immunoprecipitation analysis demonstrates enrichment of MAZ at the endogenous MCK promoter and other muscle gene promoters (Skeletal α-actin, Desmin, and α-Myosin heavy chain) in skeletal and cardiac myocytes. Consistent with its role in muscle gene transcription, MAZ transcripts and DNA-binding activity are upregulated during skeletal myocyte differentiation. Furthermore, MAZ was shown to bind numerous sequences (e.g., CTCCTCCC and CTCCACCC) that diverge from the GA box binding motif. Alternate motifs were identified in many muscle promoters, including Myogenin and MEF2C, and one motif was shown to be critical for Six4 promoter activity in both skeletal and cardiac myocytes. Interestingly, MAZ occupies and is able to transactivate the Six4 promoter in skeletal but not cardiac myocytes. Taken together, these findings are consistent with a previously unrecognized role for MAZ in muscle gene regulation.

scholarly journals A Novel PF/PN Motif Inhibits Nuclear Localization and DNA Binding Activity of the ESX1 Homeoprotein

2000 ◽  
Vol 20 (2) ◽  
pp. 661-671 ◽  
Author(s):  
Yu-Ting Yan ◽  
Stacey M. Stein ◽  
Jixiang Ding ◽  
Michael M. Shen ◽  
Cory Abate-Shen
Keyword(s):  
Dna Binding ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Binding Motif ◽  
Rich Region ◽  
Dna Binding Activity ◽  
Proline Rich Region

ABSTRACT Despite their significance for mammalian embryogenesis, the molecular mechanisms that regulate placental growth and development have not been well defined. The Esx1 homeobox gene is of particular interest because it is among the few regulatory genes that have specific expression and function in the placenta during murine development. In addition, the ESX1 protein contains several notable features that are not often associated with homeoproteins, including an atypical homeodomain of the paired-like class, a proline-rich region that contains an SH3 binding motif, and a novel repeat region consisting of prolines alternating with phenylalanines or asparagines that we term the PF/PN motif. We have found that the ESX1 protein is expressed in the labyrinth layer of the placenta in vivo, where its subcellular localization is primarily cytoplasmic. Our results suggest that this unexpected subcellular localization is conferred by the PF/PN motif, which inhibits nuclear localization of ESX1 in cell culture, as well as its DNA binding activity in vitro. Finally, we show that the proline-rich region of ESX1 mediates interactions in vitro with the c-abl SH3 domain as well as with certain WW domains. We propose that the PF/PN motif provides a novel mechanism for regulating nuclear entry and that the essential function of ESX1 during placental development is mediated by its ability to couple cytoplasmic signal transduction events with transcriptional regulation in the nucleus.

Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding activity of E2F-1/DP-1 by phosphorylation

1994 ◽  
Vol 14 (12) ◽  
pp. 8420-8431
Author(s):  
M Xu ◽  
K A Sheppard ◽  
C Y Peng ◽  
A S Yee ◽  
H Piwnica-Worms
Keyword(s):  
Dna Binding ◽  
Active Form ◽  
Cyclin A ◽  
Binding Activity ◽  
Stable Complex ◽  
Binding Studies ◽  
Dna Binding Activity ◽  
Phosphopeptide Mapping

E2F-1, a member of the E2F transcription factor family, contributes to the regulation of the G1-to-S phase transition in higher eukaryotic cells. E2F-1 forms a heterodimer with DP-1 and binds to several cell cycle regulatory proteins, including the retinoblastoma family (RB, p107, p130) and cyclin A/CDK2 complexes. We have analyzed E2F-1 phosphorylation and its interaction with cyclin A/CDK2 complexes both in vivo and in vitro. In vitro, E2F-1 formed a stable complex with cyclin A/CDK2 but not with either subunit alone. DP-1 did not interact with cyclin A, CDK2, or the cyclin A/CDK2 complex. While the complex of cyclin A/CDK2 was required for stable complex formation with E2F-1, the kinase-active form of CDK2 was not required. However, E2F-1 was phosphorylated by cyclin A/CDK2 in vitro and was phosphorylated in vivo in HeLa cells. Two-dimensional tryptic phosphopeptide mapping studies demonstrated an overlap in the phosphopeptides derived from E2F-1 labeled in vitro and in vivo, indicating that cyclin A/CDK2 may be responsible for the majority of E2F-1 phosphorylation in vivo. Furthermore, an active DNA-binding complex could be reconstituted from purified E2F-1/DP-1 and cyclin A/CDK2. Binding studies conducted both in vitro and in vivo demonstrated that the cyclin A/CDK2-binding region resided within the N-terminal 124 amino acids of E2F-1. Because the stable association of E2F-1 with cyclin A/CDK2 in vitro and in vivo did not require a DP-1- or RB-binding domain and because the interactions could be reconstituted from purified components in vitro, we conclude that the interactions between cyclin A/CDK2 and E2F-1 are direct. Finally, we report that the DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation by cyclin A/CDK2.

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

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