The Cdc6 Protein Is Ubiquitinated in Vivo for Proteolysis in Saccharomyces cerevisiae

Sánchez, Mar; Calzada, Arturo; Bueno, Avelino

doi:10.1074/jbc.274.13.9092

Cited by 60 publications

(58 citation statements)

References 39 publications

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“…There were significant differences in the distribution of studied metals in the different parts of the plants. A substantial part of all four heavy metals was accumulated in roots, as also reported by other researchers [26][27][28][29][30]. This is attributed to the fact that after penetration in the plasma, the occurring inactivation and accumulation of important amounts of heavy metals was probably a result from formation of less mobile chelation compounds with organic matter.…”

Section: Pb Zn Cu and CD Content In The Root Systemsupporting

confidence: 68%

Possibilities of wheat farming on soils contaminated with heavy metals in the region of NFMV – Plovdiv, Bulgaria

Ivanova¹,

Dospatliev²,

Stoyanova³

et al. 2017

ARTTE

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Section: Pb Zn Cu and CD Content In The Root Systemsupporting

confidence: 68%

Possibilities of wheat farming on soils contaminated with heavy metals in the region of NFMV – Plovdiv, Bulgaria

Ivanova¹,

Dospatliev²,

Stoyanova³

et al. 2017

ARTTE

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“…Although it is clear that loading of Cdc6 onto chromatin is inhibited by cyclindependent kinase (CDK) activity, the mechanism of inhibition is complex, and there are many modes of regulation (see Figure 1). Expression of CDC6 is cell cycle regulated (Zwerschke et al, 1994;Piatti et al, 1995) and therefore is at least indirectly controlled by CDK activity, such that CDC6 is expressed in late M and in G1, but is not expressed in S, G2, and early M. Phosphorylation of Cdc6 promotes ubiquitin-mediated proteolysis of Cdc6 Elsasser et al, 1999;Sanchez et al, 1999;Drury et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

Honey

Futcher

2007

MBoC

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The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of these mechanisms individually. We find that when Cdc6 is forced into the nucleus during late G1 or S, it will not substantially reload onto chromatin no matter whether its CDK sites are present or not. In contrast, at a G2/M nocodazole arrest, Cdc6 will reload onto chromatin if and only if its CDK sites have been removed. Trace amounts of nonphosphorylatable Cdc6 are dominant lethal in strains bearing nonphosphorylatable Orc2 and Orc6, apparently because of rereplication. This synthetic dominant lethality occurs even in strains with wild-type MCM genes. Nonphosphorylatable Cdc6, or Orc2 and Orc6, sensitize cells to rereplication caused by overexpression of various replication initiation proteins such as Dpb11 and Sld2.

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“…Yeast and human Cdc6 physically interact with and become phosphorylated by cyclin/Cdk complexes in vitro and in vivo (Elsasser et al, 1996(Elsasser et al, , 1999Piatti et al, 1996;Brown et al 1997;Jallepalli et al, 1997;Saha et al, 1998;Fujita et al, 1999;Jiang et al, 1999;Petersen et al, 1999;Calzada et al, 2000). Analysis of Saccharomyces cerevisiae Cdc6 and the Schizosaccharomyces pombe homologue Cdc18 revealed that phosphorylation of these proteins by Cdk targets them for ubiquitin-mediated degradation, presumably before DNA replication initiates but after the prereplicative complexes have formed Kominami and Toda, 1997;Baum et al, 1998;Elsasser et al, 1999;Sanchez et al, 1999;Drury et al, 2000). When Cdc18 degradation was inhibited by mutating the five N-terminal Cdk phosphorylation sites, an overreplication phenotype was observed, demonstrating that phosphorylation of Cdc18 by Cdk is not required to initiate but rather to prevent reinitiation of DNA replication by targeting Cdc18 for proteolytic degradation Lopez-Girona et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells

Herbig

Griffith

Fanning

2000

MBoC

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Cyclin-dependent kinases (Cdk) are essential for promoting the initiation of DNA replication, presumably by phosphorylating key regulatory proteins that are involved in triggering the G1/S transition. Human Cdc6 (HsCdc6), a protein required for initiation of DNA replication, is phosphorylated by Cdk in vitro and in vivo. Here we report that HsCdc6 with mutations at potential Cdk phosphorylation sites was poorly phosphorylated in vitro by Cdk, but retained all other biochemical activities of the wild-type protein tested. Microinjection of mutant HsCdc6 proteins into human cells blocked initiation of DNA replication or slowed S phase progression. The inhibitory effect of mutant HsCdc6 was lost at the G1/S transition, indicating that phosphorylation of HsCdc6 by Cdk is critical for a late step in initiation of DNA replication in human cells. INTRODUCTIONIn eukaryotes, the cell division cycle is regulated by the periodic activation and inactivation of cyclin-dependent kinases (Cdk), which are thought to phosphorylate protein substrates that are involved in driving cell cycle transitions (reviewed in Sherr, 1996;Roberts, 1999). The transition from G1 to S phase of the mammalian cell cycle requires the activity of cyclin E/Cdk2 and cyclin A/Cdk2. Cyclin E is expressed in middle to late G1 (Dulic et al., 1992, Koff et al., 1992, whereas cyclin A is first expressed at the G1/S transition of the cell cycle (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992). Microinjection of anticyclin E or anticyclin A antibodies into human cells or expression of antisense cyclin A RNA, inhibits initiation of DNA replication (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992;Tsai et al., 1993;Ohtsubo et al., 1995). Furthermore, enhanced levels of cyclins A and E accelerate the G1/S transition in vivo (Resnitzky et al., 1994(Resnitzky et al., , 1995 and in vitro (Krude et al., 1997).The initiation of DNA replication in eukaryotes requires the stepwise assembly of a multiprotein complex, called the prereplicative complex (pre-RC), on replicator elements in the chromatin (reviewed in Dutta and Bell, 1997;Newlon, 1997). Studies in yeast and in Xenopus have determined that a six-subunit complex, called origin recognition complex (ORC), serves to nucleate this assembly. In G1 of the cell cycle, the Cdc6 protein promotes the loading of the minichromosome maintenance proteins (MCMs) onto chromatin (Coleman et al., 1996;Aparicio et al., 1997;Donovan et al., 1997;Tanaka et al., 1997). This reaction requires an intact Walker A or Walker B motif in Cdc6 (Perkins and Diffley, 1998;Wang et al., 1999;Weinreich et al., 1999) and presumably involves direct physical interaction between Cdc6 and ORC (Li and Herskowitz, 1993;Liang et al., 1995;Mizushima et al., 2000). Although the mechanism of pre-RC assembly in human cells has not yet been characterized, all of the components of the pre-RC identified so far in yeast appear to be conserved in humans.A growing body of evidence indicates that components of the pre-RC are Cdk substrates who...

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The Cdc6 Protein Is Ubiquitinated in Vivo for Proteolysis in Saccharomyces cerevisiae

Cited by 60 publications

References 39 publications

Possibilities of wheat farming on soils contaminated with heavy metals in the region of NFMV – Plovdiv, Bulgaria

Possibilities of wheat farming on soils contaminated with heavy metals in the region of NFMV – Plovdiv, Bulgaria

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

Mutation of Cyclin/cdk Phosphorylation Sites in HsCdc6 Disrupts a Late Step in Initiation of DNA Replication in Human Cells

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