Journal of Experimental Botany, Vol. 52, No. 364, pp. 2239-2240,
November 1, 2001
© 2001 Oxford University Press
Gene Notes |
DNA replication in plants: characterization of a cdc6 homologue from Arabidopsis thaliana
1 Departamento de Bioquímica Médica, ICB, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil
2 Vakgroep Moleculaire Genética, Department Plantengenetica, VIB, Universiteit Gent, B-9000 Gent, Belgium
Received 1 June 2001; Accepted 18 June 2001
Abstract
Cdc6 is a key regulator of DNA replication in eukaryotes. In this work, the expression pattern of an Arabidopsis cdc6 homologue is characterized by RT-PCR and in situ hybridization. The data suggest that cdc6At expression is cell cycle regulated. During development, high cdc6At mRNA levels are found in regular cycling cells. In addition, cdc6At expression is also observed in cells that are probably undergoing endoreduplication, suggesting a possible role of Cdc6At in this process in plants.
Key words: Arabidopsis thaliana, CDC6, DNA replication, pre-replication complex.
The eukaryotic cell cycle is a complex and ordered mechanism to maintain the integrity and ploidy of the genome, assuring that daughter cells receive the same content of genetic material from the mother cell. In some cases, cells can have endoreduplication cycles, where they undergo rounds of DNA replication without mitosis. These events are very common in plants (Joubès and Chevalier, 2000
). Two steps are important during the control of the replication cycle: first, the formation of a pre-replication complex (pre-RC) that licenses DNA for replication; and second, the initiation of DNA replication. The pre-RC is formed by sequential assembly of a series of proteins, Origin Recognition Complex (ORC), Cdc6/Cdc18, Cdt1 and minichromosome maintenance (MCM) proteins (Stillman, 1996; Pasero and Schwob, 2000).
Cdc6/Cdc18 homologues have been identified in a number of eukaryotes. They play a central role in the regulation of DNA replication, being involved in the assembly and maintenance of the pre-RC (Stillman, 1996; Pasero and Schwob, 2000). In the absence of Cdc6/Cdc18, cells fail to initiate DNA replication, even though entering G1 (Cocker et al., 1996). cdc6/cdc18 mRNA and protein levels oscillate during the yeast cell cycle, peaking at late G1; and Cdc6 is also synthesized in late G2 (Stillman, 1996). Overexpression of Cdc6 in budding yeast does not cause endoreduplication, however overexpression of Cdc6/Cdc18 in fission yeast induces continuous DNA synthesis without mitosis, resulting in polyploid cells (Nishitani and Nurse, 1995; Muzi-Falconi et al., 1996; Wolf et al., 1999).
In order to study the controls of DNA replication in plants, a cdc6 homologue from Arabidopsis thaliana was isolated and its expression pattern was characterized.
An Arabidopsis gene encoding a protein with homology with Cdc6/Cdc18 was identified in the Arabidopsis database (Accession No. T27A16.22). The corresponding cDNA was amplified by PCR using first strand cDNA from flower buds, Pfu polymerase (Statagene), primers 5'-TTTCTGCCTCCTTCTGATAAC-3' and 5'-GAACCAACCCATCTATAAGAC-3' and annealing temperature of 45 °C. The fragment was cloned into pCR-Script AMP plasmid (Stratagene), and the nucleotide sequence was determined by automated sequencing (gb: AF275940). A second Arabidopsis cdc6 homologue, showing 69.7% identity with Cdc6aAt at amino acid level, is present in the Arabidopsis genome (Accession No. F10K1_2), but it has not been characterized so far.
cdc6At expression was analysed by RT-PCR and by in situ hybridization. Total RNA from etiolated plants, roots, stems, leaves, siliques, and flower buds of in vitro growing Arabidopsis plants ecotype C24 was extracted (Logemann et al., 1987). Sterile root cultures were established by culturing 3-week-old in vitro growing Arabidosis seedlings in liquid Murashige and Skoog medium with 3% sucrose at 22 °C, with constant agitation. Root cultures were treated with 30 µM oryzalin or 10 mM hydroxyurea in the same medium and conditions, for 48 h. Etiolated seedlings were obtained by germinating seeds in the dark for 10 d. First strand cDNAs of the distinct RNAs were synthesized (First strand cDNA Synthesis kit, Pharmacia Biotech). PCR was carried out using primers 5'-GAACCAACCCATCTATAAGAC-3' and 5'-TTGACACCATCCAGTCTCTACCTC-3', specific for cdc6At amplification; primers 5'-ATGCAGATCTTCGTTAAGACT-3' and 5'-ACCACC-ACGGAGCCTGAGAAC-3' for ubiquitin amplification; primers 5'-ATGTCGGGTCGTGGAAAGGGAG-3' and 5'-CTTAACCGCCGAATCCGTAACC-3' for histone H4 amplification and primers 5'-TGG-AGAATATGGTGCACTATTTGGC-3' and 5'-GAAACTGACTTCTTACAAGATATGC-3' for Arath, cycB1;1 amplification.
The amplified products were transferred to nylon membrane and hybridized with cdc6At, ubiquitin, histone H4, and cycB1;1 cDNAs as probes. In situ hybridization was performed essentially as described previously (de Almeida Engler et al., 2001). Slides were hybridized with 35S-labelled cdc6At antisense and sense RNA, as probes.
In order to study the controls of DNA replication in plants, an Arabidopsis cdc6 cDNA homologue was isolated and characterized. cdc6At has an open reading frame of 539 amino acids encoding a putative protein of 59.9 kDa (Fig. 1). Amino acid sequence comparison between Cdc6At and its homologues HCdc6, XCdc6, SpCdc18, and ScCdc6 showed 27%, 29%, 25%, and 24% of identity, respectively. The deduced amino acid sequence contains a region of 272 residues that is conserved between all Cdc6 homologues. It corresponds to the CDC-NTP domain, which has two-peptide motifs known as Walker-A and B motifs 73 (159GCPGTGKS and 249DEMD, respectively), common to nucleotide binding proteins 74 (Wang et al., 1999). It has been demonstrated that Cdc6 can be regulated by phosphorylation, which is responsible for targeting Cdc6 for ubiquitination and proteolysis 76 (Elsasser et al., 1999). Cdc6At contains three consensus CDK phosphorylation sites in the N-terminal region, 10SPQK, 60TPMK and 66SPRR, suggesting that this plant homologue is also regulated by phosphorylation events. A conserved nuclear localization signal, 60RKRK63 in the N-terminal portion is present in Cdc6At and in its homologues (Takei et al., 1999).
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The cdc6At expression during the plant cell cycle was investigated in roots treated with the cell cycle blockers hydroxyurea and oryzalin, that arrest cells at S phase and G2/M boundary, respectively (Verhoeven et al., 1990
). Figure 2A shows that cdc6At expression increases in roots after treatment with 30 µM oryzalin, and it is drastically reduced in roots after treatment with 10 mM HU. As expected, levels of histone H4 mRNA, an S-phase marker, are lower in the roots treated with oryzalin; and cycB1;1 expression, preferential in G2/M transition of dividing cells (Ferreira et al., 1994), decreases significantly in the roots treated with HU. Taken together, the data suggest that expression of the Arabidopsis cdc6 homologue is cell cycle regulated, and that a transcriptional burst possibly occurs in late G2, as described for S. cerevisae cdc6 (Piatti et al., 1995), and that transcription is probably over before cells enter S phase.
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cdc6At expression in different plant organs and etiolated plants was also analysed. RT-PCR analysis revealed higher levels of cdc6At mRNA in roots and etiolated plants (Fig. 2B
). In situ hybridization analysis showed that cdc6At is strongly expressed all over the hypocotyl of etiolated plants (Fig. 2C, upper panel) and in root tissues of the vascular cylinder and sites of secondary root initiation (data not shown). Interestingly, a patchy hybridization pattern of cdc6At is also observed scattered throughout mature leaves (Fig. 2C, lower panel). The expression profile of cycB1;1 did not correlate with that of cdc6At in roots and etiolated plants (Fig. 2B), suggesting that cdc6At expression is not exclusive to dividing cells. It has been demonstrated that Arabidopsis etiolated plants undergo rounds of DNA replication without mitotic cycle in the hypocotyl (Gendreau et al., 1997). Endoreduplication has also been described in Arabidopsis leaf tissues and in parenchyma and cortex root cells of various species (Joubès and Chevalier, 2000). Altogether, the data correlates cdc6At expression with typical cycling cells; and possibly also with endoreduplicating cells. Therefore, in plants, Cdc6At could be involved in licensing DNA for replication both in a regular cell cycle and in endoreduplication cycles.
Acknowledgments
The authors thank Luíza da Silva and Sônia Cristina Freire Silva for technical assistance. ASH and PCGF are indebted to the Conselho Nacional de Desenvolvimento Científico e Tecnológico 112 (CNPq). GBAR was a recipient of a CAPES fellowship.
Notes
3 To whom correspondence should be addressed. Fax: +55 21 2708647. E-mail: hemerly{at}bioqmed.ufrj.br 
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