Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing.
All organisms need to ensure that no DNA segments are rereplicated in a single cell cycle. Eukaryotes achieve this through a process called origin licensing, which involves tight spatiotemporal control of the assembly of prereplicative complexes (pre-RCs) onto chromatin. Cdt1 is a key component and crucial regulator of pre-RC assembly. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent DNA rereplication. Here, we address the mechanism of DNA licensing inhibition by Geminin, by combining X-ray crystallography, small-angle X-ray scattering, and functional studies in Xenopus and mammalian cells. Our findings show that the Cdt1:Geminin complex can exist in two distinct forms, a "permissive" heterotrimer and an "inhibitory" heterohexamer. Specific Cdt1 residues, buried in the heterohexamer, are important for licensing. We postulate that the transition between the heterotrimer and the heterohexamer represents a molecular switch between licensing-competent and licensing-defective states.
De Marco
V
V
Department of Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.
Gillespie
P J
PJ
Li
A
A
Karantzelis
N
N
Christodoulou
E
E
Klompmaker
R
R
van Gerwen
S
S
Fish
A
A
Petoukhov
M V
MV
Iliou
M S
MS
Lygerou
Z
Z
Medema
R H
RH
Blow
J J
JJ
Svergun
D I
DI
Taraviras
S
S
Perrakis
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D018360:Crystallography, X-Ray; D004261:DNA Replication; D064248:Geminin; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D016415:Sequence Alignment; D014961:X-Ray Diffraction; D014982:Xenopus laevis
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; C577995:GMNN protein, human; D064248:Geminin; D011993:Recombinant Fusion Proteins