Ferenbach A et al. (2005), Functional domains of the Xenopus replication l...

XB-ART-2466

Nucleic Acids Res 2005 Jan 12;331:316-24. doi: 10.1093/nar/gki176.

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Functional domains of the Xenopus replication licensing factor Cdt1.

Ferenbach A , Li A , Brito-Martins M , Blow JJ .

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During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition.

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Species referenced: Xenopus
Genes referenced: cdt1 gmnn mcm2 mcm6.2 mmut

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	Figure 1. Summary of constructs used in this paper. The indicated regions of Xenopus Cdt1 were expressed in E.coli as GST fusions. The two regions predicted to form coiled-coils are indicated with cross hatching. Where deletions are predicted to significantly disrupt coiled-coil formation, the cross-hatching is replaced by diagonal shading. Results for the assays outlined in subsequent figures are summarized on the right. Data not explicitly shown in this paper are indicated by asterisk.
	Figure 2. Licensing assay. (A) Interphase Xenopus egg extract or Cdt1-depleted extract was supplemented with Xenopus sperm nuclei (10 ng DNA/Î¼l) and different GST-Cdt1 constructs at 5 nM. After incubation for 30 min, chromatin was isolated and immunoblotted for the presence of Mcm2 and Mcm6. (B) Cdt1-depleted extract was supplemented with Xenopus sperm nuclei (11.5 ng DNA/Î¼l) and different GST-Cdt1 constructs at 42 nM (diagonal shading). After incubation for 30 min, aliquots were supplemented with 2.8 vol of Xenopus egg extract supplemented with [Î±-32P]dATP and 60 nM geminin. After 90 min, total DNA synthesis was assessed by TCA precipitation. As controls for the sufficiency of geminin inhibition, assays were also performed, where the Cdt1-depleted extract was replaced with buffer (white boxes). The right-hand panel also shows the activity of two His-tagged Cdt1 constructs (243â620 and 193â447).
	Figure 3. Mcm2-7 binding assay. (A) Coomassie-stained gel of purified Mcm2-7 used in the binding assay. The migration of molecular weight markers is shown to the right (size in kDa). (B) GlutathioneâSepharose beads were incubated for 30 min with 5 nM purified Mcm(2,4,6,7) and 140 nM GST-tagged Cdt1 constructs. After washing, bead-bound protein was eluted and immunoblotted for the presence of Mcm6.
	Figure 4. Overlay (far-western) assay. (A and B) Different GST-Cdt1 constructs were separated by SDSâPAGE. (A) Gels were stained with Coomassie. Molecular weight markers (sizes in kDa) are shown to the left. (B) Proteins were blotted onto nitrocellulose. After blocking, the nitrocellulose was incubated with recombinant gemininDEL, followed by anti-geminin antibody and then anti-rabbit secondary antibody coupled to horseradish peroxidase. Bound geminin was revealed by ECL chemiluminescence. To give a comparable signal, Cdt1(1â620) was used at 37-fold lower concentration than the other fragments and was not visible by Coomassie staining; the position of the protein is indicated by a dashed oval. (C) His-tagged Cdt1(193â447) and an S-tagged version of geminin capable of binding Cdt1 (mini-geminin) were expressed together and separately in E.coli. After protein expression, cells were lysed and the proteins separated into soluble and insoluble components. Soluble protein was applied to nickel-NTA columns; the flow-through was collected, and then bound protein was eluted with imidazole. Fractions were separated by SDSâPAGE and immunoblotted for Cdt1 and geminin.
	Figure 5. Geminin neutralization. (A) Xenopus egg extract was supplemented with sperm nuclei (3ng DNA/Î¼l), 60 nM gemininDEL, [Î±-32P]dATP and 50 nM GST-Cdt1 constructs. After incubation for 90 min, total DNA synthesis was assessed by TCA precipitation. Asterisked columns show constructs shown to possess licensing activity. (B) Xenopus egg extract was supplemented with sperm nuclei (3 ng DNA/Î¼l), [Î±-32P]dATP, various concentrations of gemininDEL and either 6.5 nM His-Cdt1(1â620) (black circles), 7.1 nM His-Cdt1(243â620) (black squares), 7.7 nM His-Cdt1(193â447) (white triangles) or buffer alone (white circles). After incubation for 90 min, total DNA synthesis was assessed by TCA precipitation.
	Figure 6. Characterization of complexes between Cdt1(193â447) and mini-geminin. Recombinant complexes between His-Cdt1(193â447) and His-tagged mini-geminin were prepared. (A and B) Complexes were subject to gel filtration. (A) Fractions were subjected to SDSâPAGE and immunoblotted for Cdt1 and geminin. The migration of molecular weight markers (sizes in kDa) is also shown. (B) The peak fraction of geminin and Cdt1 from the gel filtration column was immunoprecipitated with either anti-geminin antibodies or non-immune antibodies; precipitates were then immunoblotted for Cdt1 and geminin. (C) Complexes were subject to glycerol gradient sedimentation. Fractions were subjected to SDSâPAGE and immunoblotted for Cdt1 and geminin. The migration of molecular weight markers (sizes in kDa) is also shown. (D) Different quantities of the complex were subjected to SDSâPAGE and stained with Coomassie.
	Figure 7. Model of Cdt1 domains. (A) Schematic diagram showing the minimum domains necessary for Mcm binding, licensing activity and geminin binding. Predicted coiled-coil domains are shown with hatching. (B) Diagram of the structure of the Cdt1-geminin interface redrawn from reference (22). The fragment of Cdt1 corresponds to Xenopus Cdt1 residues 232â426. The proposed alignments of Cdt1 residues 193â231, which are predicted to form a coiled-coil, are shown as a dashed line. The coiled-coil section of geminin is shown with heavy lines.

References [+] :

Ballabeni, Human geminin promotes pre-RC formation and DNA replication by stabilizing CDT1 in mitosis. 2004, Pubmed, Xenbase