Abstract
Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins.
Highlights
Cdt1 is an essential component for the assembly of a pre-replicative complex
Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi
Expression and Purification of Recombinant Proteins in E. coli— FLAG- and His6-tagged mouse Cdt1, mouse Mcm6, and C. elegans CDT-1, GST-tagged C. elegans NOB-1 and CEH-32, and T7 as well as His6-tagged mouse geminin and C. elegans GMN-1 were overproduced in the E. coli strain BL21(DE3) and purified as described [8]
Summary
Cloning of C. elegans cdt-1, mcm-6, and gmn-1 cDNAs—The amino acid sequence of Drosophila geminin was used to screen the NCBI data base for C. elegans geminin, which yielded a sequence with the accession number Y75B8A.17. The cdt-1 and mcm-6 PCR products were digested with NdeI-XhoI and inserted into NdeI- and XhoI-digested pET24b-FLAG, which contains the FLAG tag sequence in the 5Ј-region as described previously [8]. CDNAs for C. elegans cdt-1, mcm-6, gmn-1, and orc-2 (yk1265f03 and yk236f8) were amplified using specific-sequence primers with appropriate restriction sites. Expression and Purification of Recombinant Proteins in E. coli— FLAG- and His6-tagged mouse Cdt, mouse Mcm, and C. elegans CDT-1, GST-tagged C. elegans NOB-1 and CEH-32, and T7 as well as His6-tagged mouse geminin and C. elegans GMN-1 were overproduced in the E. coli strain BL21(DE3) and purified as described [8]. The licensing reaction was carried out by a 15-min incubation of a reaction mixture (4 l) containing competent chromatin, partially purified Mcm fraction, and GST-fused Xenopus Cdt in the absence or presence of 50 ng lϪ1 Xenopus geminin or C. elegans GMN-1. Delivery of dsRNA into worms was performed by soaking [26] or microinjection [27]
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