Disease relevance of CDC6

• The CDC6 gene was expressed in Escherichia coli as a glutathione-S-transferase fusion protein and purified by affinity chromatography [1].
• Furthermore, human lung carcinomas with high levels of Cdc6 are associated with low levels of p16(INK4a) [2].

High impact information on CDC6

• We have cloned a Xenopus Cdc6 homolog (Xcdc6) and characterized its role in DNA replication with Xenopus egg extracts [3].
• Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae [4].
• Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation [4].
• Here we show that B-type CDKs in Saccharomyces cerevisiae prevent re-initiation through multiple overlapping mechanisms, including phosphorylation of the origin recognition complex (ORC), downregulation of Cdc6 activity, and nuclear exclusion of the Mcm2-7 complex [5].
• Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway [6].

Biological context of CDC6

• The Saccharomyces cerevisiae gene CDC6, whose protein product is required for DNA replication, is transcribed only in late G1 and S phases [7].
• The CDC6 gene is normally transcribed at the end of mitosis, but in cells with a prolonged G1 phase there is a second burst of transcription in late G1 [8].
• Thus CDC6 appears to have an important and perhaps unique dual role in S phase, it is first required for the initiation of DNA replication and then actively participates in the suppression of nuclear division [7].
• Dual functions of CDC6: a yeast protein required for DNA replication also inhibits nuclear division [7].
• We have compared the periodic fluctuation of mRNAs encoded by CDC6, a cell cycle gene controlling initiation of DNA replication, and CLN1, a G1 cyclin gene expressed at late G1 [1].

Anatomical context of CDC6

• Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins [9].
• A plasmid-encoded CDC6-beta-galactosidase hybrid protein was located at the plasma membrane by indirect immunofluorescence [10].
• CDC6 requirement for spindle formation during maturation of mouse oocytes [11].

Associations of CDC6 with chemical compounds

• We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6 temperature-sensitive mutant cells [12].
• DNA sequence analysis of the CDC6 promoter revealed two sequences, 5'-C/GACGCGNC/G-3' and 5'-PuGNAGAAA-3' (where Pu is a purine, and N is any nucleotide), which are repeated three times each [13].
• Unlike histone H2A mRNA, the CDC6 mRNA as well as CDC8 mRNA were not affected by hydroxyurea treatment [13].
• The amino acid sequence of the 48 kDalton CDC6 gene product, as deduced from DNA sequence data, includes the three consensus peptide motifs involved in guanine nucleotide binding and GTPase activity, a target site for cAMP-dependent protein kinase and a carboxy-terminal domain related to metallothionein sequences [10].
• In contrast, at a G2/M nocodazole arrest, Cdc6 will reload onto chromatin if and only if its CDK sites have been removed [14].

Physical interactions of CDC6

• Indeed, Mcm7 stimulates Mcm1 binding to the early cell cycle box upstream of the promoters of MCM7 as well as CDC6 and MCM5 [15].
• Inhibiting Cdc6 ATP hydrolysis stabilizes Cdt1 on origin DNA and prevents Mcm2-7 loading [16].
• Strikingly, Cdc6 interacts preferentially with B-type cyclin/Cdc28 complexes and not Cln/Cdc28 in log-phase cells [17].
• As distamycin A is a specific reagent that binds noncovalently to DNA at (A + T)-rich tracks, the stimulation of Abf1 DNA binding activity may be mediated by the Cdc6/DNA interaction [18].
• The Cdc6 protein interacts in vivo with Cdc2 kinase complexes [19].

Enzymatic interactions of CDC6

• Indeed, we have found that Cdc6 is ubiquitinated in vivo and degraded by a Cdc4-dependent mechanism [20].

Regulatory relationships of CDC6

• We provide evidence that the replication defect of mcm1 mutants can be suppressed by ectopic CDC6 transcription [21].
• Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts [22].
• This inhibition requires that Clb2 be induced before Cdc6, an initiation protein required for pre-RC formation; once pre-RCs have formed, Clb2 can no longer inhibit initiation [23].
• Saccharomyces cerevisiae Cdc6 stimulates Abf1 DNA binding activity [18].

Other interactions of CDC6

• Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once C1b/Cdk1 kinases have been activated [24].
• However, in cdc6 and cdc14 mutants, the mitotic loss of pDK243 with an ARS cluster was suppressed by a factor of 6-8 compared to pDK243 without the cluster [25].
• To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo [12].
• To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins [12].
• Introducing a copy of the wild-type CDC6 gene into the cdc6-1 mutant strain restored the Abf1 DNA binding activity [18].

Analytical, diagnostic and therapeutic context of CDC6

• CDC6 mRNA accumulates in cdc15-arrested telophase cells, decays around cell separation, and reappears during telophase and nuclear division of the next cycle [1].
• Northern blotting shows that the size of the CDC6 mRNA is about 1.7 kilobases [26].
• Molecular cloning of Saccharomyces cerevisiae CDC6 gene. Isolation, identification, and sequence analysis [26].
• A Southern blot of yeast chromosomes which were separated by the field inversion gel electrophoresis method indicates that the isolated DNA fragment is derived from chromosome X. The locus from which the clone was derived was marked by integration with a nutritional marker and found by meiotic mapping to cosegregate with CDC6 [26].
• Using the yeast two-hybrid system and immunoprecipitation, we report here that murine HP1alpha interacts with the preRC proteins ORC1, ORC2 and CDC6 [27].

References