Mouse geminin inhibits not only Cdt1-MCM6 interactions but also a novel intrinsic Cdt1 DNA binding activity.
DNA replication is controlled by the stepwise assembly of a pre-replicative complex and the replication apparatus. Cdt1 is a novel component of the pre-replicative complex and plays a role in loading the minichromosome maintenance (MCM) 2-7 complex onto chromatin. Cdt1 activity is inhibited by geminin, which is essential for the G(2)/M transition in metazoan cells. To understand the molecular basis of the Cdt1-geminin regulatory mechanism in mammalian cells, we cloned and expressed the mouse Cdt1 homologue cDNA in bacterial cells and purified mouse Cdt1 to near homogeneity. We found by yeast two-hybrid analysis that mouse Cdt1 associates with geminin, MCM6, and origin recognition complex 2. MCM6 interacts with the Cdt1 carboxyl-terminal region (amino acids 407-477), which is conserved among eukaryotes, whereas geminin associates with the Cdt1 central region (amino acids 177-380), which is conserved only in metazoans. In addition, we found that Cdt1 can bind DNA in a sequence-, strand-, and conformation-independent manner. The Cdt1 DNA binding domain overlaps with the geminin binding domain, and the binding of Cdt1 to DNA is inhibited by geminin. Taken together, we have defined structural domains and novel biochemical properties for mouse Cdt1 that suggest that Cdt1 behaves as an intrinsic DNA binding factor in the pre-replicative complex.
Yanagi
Ken-ichiro
K
Cellular Physiology Laboratory, RIKEN (The Institute of Physical and Chemical Research) and CREST, Japan Science and Technology Corporation, Wako, Saitama 351-0198, Japan.
Mizuno
Takeshi
T
You
Zhiying
Z
Hanaoka
Fumio
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D064248:Geminin; D006801:Humans; D051379:Mice; D064151:Minichromosome Maintenance Complex Component 6; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011233:Precipitin Tests; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C577995:GMNN protein, human; D064248:Geminin; C577997:Gmnn protein, mouse; D009687:Nuclear Proteins; C103258:MCM6 protein, human; C491205:Mcm6 protein, mouse; D064151:Minichromosome Maintenance Complex Component 6