'pre-replicative complex' (pre-RC) assembles, which is characterized by an additional region of protection adjacent The budding yeast Cdc6 protein (Cdc6p) is essential to the ORC binding site . This is for formation of pre-replicative complexes (pre-RCs) approximately the time when Cdc6p first appears (Piatti at origins of DNA replication. Regulation of pre- RC et al., 1995 bound to chromatin during G 1 (Donovan et al., 1997) and Cdc6p is normally only present at high levels during pre-RCs are thermolabile in vivo in a cdc6 temperaturethe G 1 phase of the cell cycle. This is partly because sensitive mutant Detweiler and the CDC6 gene is only transcribed during G 1 . In this Li, 1997). CDC6 interacts genetically with the origin article we show that rapid degradation of Cdc6p also recognition complex (ORC) (Li and Herskowitz, 1993; contributes to this periodicity. Cdc6p degradation rates Liang et al., 1995;Loo et al., 1995) In both fission and budding yeasts cyclin-dependent longer degraded by the Cdc4/34/53 pathway is, none protein kinases (cdks) play a key role in blocking rethe less, fully functional. Constitutive overexpression replication (Broek et al., 1991;Hayles et al., 1994; Moreno of either wild-type or stable Cdc6p does not induce re Dahmann et al., 1995;Piatti et al., replication and does not induce assembly of pre-1996). This occurs in budding yeast, at least in part, replicative complexes after DNA replication is because Cdc28p (cdk1), together with the B-type cyclins, complete.blocks formation of pre-RCs. Expression of Cdc6p during Keywords: cell cycle/DNA replication/proteolysis G 1 can promote efficient formation of pre-RCs and subsequent DNA replication. However, Cdc6p expressed after a 'point of no return' in late G 1 can no longer promote pre-RC formation and DNA replication (Piatti et al.,
Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase. The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing.
Expression of the Cdc6 protein (Cdc6p) is essential for formation of prereplicative complexes at budding yeast replication origins. Analysis of mutations in the conserved nucleoside triphosphate (NTP)-binding site of Cdc6p described here suggests that NTPs are required both for the productive interaction of Cdc6p with replication origins during G1 and the quantitative loading of the Mcm2-7 family of proteins onto chromatin. We show that Cdc6p exhibits significant sequence similarity to subunits of eukaryotic and prokaryotic clamp-loaders, which load ring-shaped DNA polymerase processivity factors onto DNA in an analogous reaction. Similarities in both sequence and mechanism suggest that Cdc6p and the clamp-loaders are members of a superfamily of nucleotide-dependent loading factors.
G.Perkins and L.S.Drury contributed equally to this paperThe Cdc6 DNA replication initiation factor is targeted for ubiquitin-mediated proteolysis by the E3 ubiquitin ligase SCF CDC4 from the end of G 1 phase until mitosis in the budding yeast Saccharomyces cerevisiae. Here we describe a dominant-negative CDC6 mutant that, when overexpressed, arrests the cell cycle by inhibiting cyclin-dependent kinases (CDKs) and, thus, prevents passage through mitosis. This mutant protein inhibits CDKs more ef®ciently than wild-type Cdc6, in part because it is completely refractory to SCF CDC4 -mediated proteolysis late in the cell cycle and consequently accumulates to high levels. The mutation responsible for this phenotype destroys a putative CDK phosphorylation site near the middle of the Cdc6 primary amino acid sequence. We show that this site lies within a novel Cdc4-interacting domain distinct from a Cdc4-interacting site identi®ed previously near the N-terminus of the protein. We show that both sites can target Cdc6 for proteolysis in late G 1 /early S phase whilst only the newly identi®ed site can target Cdc6 for proteolysis during mitosis.
GATA-2 is a member of a family of transcription factors which bind a common DNA sequence motif (WGA-TAR) through an evolutionarily conserved zinc finger domain. An essential role for GATA-2 in the development of hematopoietic stem cells has recently been shown in gene targeting experiments in mice. Here we show that GATA-2 exists in hematopoietic progenitor cells as a phosphoprotein. Stimulation of progenitors with interleukin-3 (IL-3) results in enhanced phosphorylation of GATA-2 which occurs within 5 min. IL-3 is known to signal in part through mitogen-activated protein (MAP) kinase, and evidence for MAP kinase signaling in the control of GATA-2 phosphorylation was obtained by genetically manipulating the MAP kinase pathway in COS cells using either constitutively activating or interfering mutants of MAP kinase kinase. Furthermore, using an interfering mutant of MAP kinase kinase, we directly demonstrated a critical role for the MAP kinase pathway in the IL-3-dependent phosphorylation of GATA-2 in hematopoietic progenitor cells. Finally, in vitro phosphorylation experiments using recombinant GATA-2 raise the possibility that MAP kinase itself may phosphorylate GATA-2. Our results provide evidence for phosphorylation via the MAP kinase pathway constituting a cytoplasmic link between GATA-2 and growth factor receptors and are consistent with the hypothesis that GATA-2 is involved in the growth factor responsiveness and proliferation control of hematopoietic progenitor cells.
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