title: Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex.
abstract: All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.
authors.affiliation: a Department of Genome Medicine , Tokyo Metropolitan Institute of Medical Science , Tokyo , Japan.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: You
authors.affiliation: b Department of Biological Sciences , Graduate School of Science, Osaka University , Toyonaka , Osaka , Japan.
authors.forename: Koji L
authors.initials: KL
authors.lastname: Ode
authors.affiliation: c Laboratory of Protein Analysis, Tokyo Metropolitan Institute of Medical Science , Tokyo , Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Shindo
authors.affiliation: b Department of Biological Sciences , Graduate School of Science, Osaka University , Toyonaka , Osaka , Japan.
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Takisawa
authors.affiliation: a Department of Genome Medicine , Tokyo Metropolitan Institute of Medical Science , Tokyo , Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D064248:Geminin / Q000378:metabolism*; D051379:Mice; D064110:Minichromosome Maintenance Proteins / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C406819:Cdt1 protein, Xenopus; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C577998:GMNN protein, Xenopus; D064248:Geminin; D050505:Mutant Proteins; D011994:Recombinant Proteins; C498645:Ris2 protein, mouse; D029867:Xenopus Proteins; D004247:DNA; D001120:Arginine; D004265:DNA Helicases; D064110:Minichromosome Maintenance Proteins
keywords: Cdc2/Cyclin B; Cdc7 kinase; Cdk2/Cyclin A; Cdt1; DNA helicase; DNA replication; DNA-binding activity; Mcm; cell cycle; licensing