1. Volume 15, Issue 2, Pages 245-258 (July 2004)
A Dimerized Coiled-Coil Domain and an Adjoining Part of Geminin Interact with Two Sites on Cdt1 for Replication Inhibition 
Sandeep Saxena, Ping Yuan, Suman Kumar Dhar, Takeshi Senga, David Takeda, Howard Robinson, Sally Kornbluth, Kunchithapadam Swaminathan, Anindya Dutta 
Molecular Cell 
Volume 15, Issue 2, Pages (July 2004)
DOI: /j.molcel

2. Figure 1 Residues 93–152 of Geminin Are Sufficient to Bind to Cdt1
(A) Schematic diagram of geminin wild-type and mutants. The black box indicates the coiled-coil domain of geminin (Gem) predicted by the COILS program. The amino acid residues delimiting the predicted coiled-coil domain are marked. Asterisks indicate E residues changed to alanines while open triangles indicate mutations of K to A.
(B) Minimal region of geminin required for Cdt1 interaction in vitro. Pull-down of His6-geminin WT or mutants on GST-Cdt1 beads followed by SDS-PAGE of bound proteins and immunoblot with anti-His antibodies. The input lanes are shown on the left. The forms of geminin used for this experiment were WT, , , N-116, and Right panel shows the presence of GST-Cdt1 in all the lanes.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Structure of Geminin from 92–152
(A) Upper panel: the structure of the Cdt1 interacting domain of geminin, residues The two long α helices of the coiled-coil are colored in green and blue. The termini are marked. Lower panel: amino acid sequence of coiled-coil domain of geminin. The heptad repeats of coiled-coil domain are enclosed in rectangles. The residues of the heptad repeat are labeled from a to g.
(B) A helical wheel representation of the geminin coiled-coil sequence, highlighting the a and d positions at the center of the dimer interface. The positions of the residues of the heptad repeat are indicated. Salt bridges between the two chains are represented by dashed lines. Positively charged residues are in blue while negatively charged residues are in red.
(C) A portion of the 2Fo-Fc electron density map, contoured at the 1.0 σ level. Residues are marked.
(D) Surface representation of geminin monomer by electrostatic potential. (Left) and (Right) are front and back views of a single α-helix. The dark red region indicates a potential of < -10 kT/e, while the dark blue indicates > 10 kT/e. The electrostatic potentials were calculated by GRASP (Nicholls et al., 1991).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Geminin Dimerizes through the Coiled-Coil Domain and the Dimerization Is Critical for the Binding to Cdt1
(A) Gel filtration of geminin WT and geminin LZ. Fractions subjected to SDS-PAGE and immunoblot with anti-His antibody. Elution fractions of molecular mass standards are indicated at the top.
(B) Glycerol gradient analysis of geminin WT and geminin LZ. Alternate fractions collected from the bottom followed by SDS-PAGE and Western blot analysis. Fractions containing the peaks of molecular mass markers are shown at the top.
(C) Geminin dimerization requires the hydrophobic residues of the coiled-coil domain but not the surface E residues: 293T cells were cotransfected with plasmids expressing HA-geminin WT or HA-geminin EA and GST-geminin WT, GST-geminin LZ, or GST-geminin EA followed by GST pull-down. Immunoblot of input (I) and pulled down (P) proteins with indicated antibodies.
(D) Intact coiled-coil domain of geminin is required for Cdt1 interaction in vitro: pull-down of His6-geminin WT or LZ mutant on GST-Cdt1 beads followed by SDS-PAGE of bound proteins and immunoblot with anti-His antibodies.
(E) In vivo interaction of Cdt1 with geminin WT or geminin LZ: 293T cells were transfected with HA-Cdt1 and GST-geminin WT or GST-geminin LZ. Input lanes show the expression of all the proteins whereas the pull-down lanes show proteins associated with glutathione agarose beads. Top, immunoblot with anti-HA; bottom, immunoblot with anti-GST.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 Bipartite Interactions between Geminin and Cdt1
(A) Cdt binds to surface glutamic acid residues of the coiled-coil domain of geminin. GST-Cdt1 or GST-Cdt was used to pull-down His6-geminin WT, E1A, E2A, EA, or KA. Proteins associated with GST beads were visualized by immunoblotting with anti-geminin.
(B) Cdt1 full length has a second binding site on portion of geminin. His6-geminin 1-152, , and either WT or EA mutations were pulled down on GST-Cdt1 and visualized by immunoblotting with anti-geminin antibody.
(C) Binding interaction of the coiled-coil domain of geminin is more sensitive to salt concentrations than the N-terminal interactions. GST-Cdt1 was used to pull-down His6-geminin (lanes 1, 3, and 5) or His6-geminin (lanes 2, 4, and 6) in the presence of salt concentrations indicated at the top. Input of His6-geminin (lane7) and His6-geminin (lane 8) are shown. Proteins associated with GST beads were visualized by immunoblotting with anti-geminin. Bottom panel shows the presence of GST-Cdt1 in all the lanes.
(D) N terminus of Cdt1 can interact independently with region of geminin. His6-geminin EA was pulled down on full-length and truncated forms of GST-Cdt1 and detected by immunoblotting with anti-geminin antibody.
(E) Cdt1 provides the domain for interaction with the coiled-coil region of geminin: His6-geminin (lanes 1–13) and His6-geminin EA (lanes 14–16) were pulled down on full-length and mutant forms of GST-Cdt1 and detected by immunoblotting with anti-geminin antibody.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Disruption of Either Part of the Bipartite Interaction between Geminin and Cdt1 Diminish the Inhibitory Effect of Geminin on Cdt1 In Vitro
(A) Geminin LZ, Δ and -EA do not inhibit replication in Xenopus egg extracts: Xenopus egg extracts were incubated with 200 ng of geminin WT or mutants followed by the addition of sperm chromatin and α32PdATP. The reaction mixtures were incubated at 23°C before the addition of stop buffer. Asterisk indicates the replication products. Lane numbers for different geminin mutants have been marked.
(B) Immunoblotting with anti-His antibody to detect geminin used for replication inhibition.
(C) Titration of geminin amount for inhibition of replication in Xenopus egg extracts. Amount of replication was quantified using phosphorimager.
(D) Mutations in Cdt1 that affect geminin binding at either of the two interacting domains prevent inhibition by geminin. Replication inhibition observed with 85 ng geminin was rescued with different amount of wild-type or mutant Cdt1. Extent of replication is represented as percent of maximum rescue of replication observed in the experiment.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Two Parts of Geminin Are Required for the Inhibition of Cdt1 In Vivo and for Binding to HoxA11
(A) Geminin , Δ 70-92, LZ, and - EA do not inhibit replication of oriP based episomes. Southern blot to detect DpnI resistant p367 DNA that has replicated in HCT116 cells following cotransfection of 1μg of p367 and 6 μg of a plasmid expressing GST (pEBG), and indicated GST-geminin fusion proteins. Lanes 1 and 2 contain p367 mixed with mock transfected HCT116 DNA. Arrow indicates DpnI resistant DNA in lanes 3 onward. Lane numbers for different mutants of geminin have been marked.
(B) Equivalent expression of different proteins used in (A). Transfected cell lysates after SDS-PAGE and anti-GST Western blot.
(C) Model of the interaction of geminin with Cdt1. E, glutamates on the surface of geminin mutated in geminin EA mutant. Dashed curve, the segment of geminin that is unstructured in the crystal structure and may make a stable conformation when complexed with Cdt1. The dashed lines between the geminin subunits indicate atypical intersubunit interactions. The N-100 and region of Cdt1 are indicated. The fact that E1A and E2A can both interact with and inhibit Cdt1 suggests that there are at least two ways in which Cdt1 can associate with the geminin cylinder. The flexibility between the two forms could be due to flexibility between the two domains of Cdt1 (as shown) or between the region and coiled-coil of geminin.
(D) Surface E residues of coiled-coil domain of geminin are required for interaction with HoxA11. Bottom, S35 methionine labeled HoxA11 was pulled down on His6-geminin WT (lane 1) or EA (lane 2) beads and exposed to phosphorimager. Top, Coomassie staining shows amount of bound geminin protein.
(E) HoxA11 does not have a secondary binding interaction with region of geminin. Bottom, S35 labeled HoxA11 was pulled down on different His6-geminin proteins. Top, Coomassie staining shows amount of bound geminin protein. Lane 1, WT geminin; lane 2, Geminin ; lane 3, Geminin
Molecular Cell  , DOI: ( /j.molcel )