Xenopus Cdc6 performs separate functions in initiating DNA replication.
Cdc6 performs an essential role in the initiation of eukaryotic DNA replication by recruiting the minichromosome maintenance (MCM) complex onto DNA. Using immunodepletion/add-back experiments in Xenopus egg extracts, we have determined that both Walker A (ATP binding) and Walker B (ATP hydrolysis) motifs of Xenopus Cdc6 (Xcdc6) are essential, but have distinct functional roles. Although Walker B mutant protein binds chromatin well, Walker A mutant protein binds chromatin poorly. Neither Walker A nor Walker B mutant protein, however, load appreciable MCM onto DNA. Herein, we provide evidence that Cdc6 functions as a multimer: 1) mutant and wild-type Xcdc6 form multimers; 2) either mutant protein is dominant negative when added before wild-type Xcdc6, but stimulates DNA replication when added simultaneously with wild-type Xcdc6; and 3) the two mutants restore DNA replication when added together, in the absence of wild-type Xcdc6. Our findings suggest that ATP may play a key regulatory role within this multimer: its binding to Cdc6 promotes chromatin association and its hydrolysis facilitates MCM loading. Moreover, ATP binding and hydrolysis may occur in trans between Cdc6 subunits within the complex.
Frolova
Natalya S
NS
Molecular Oncology Program, Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Schek
Nancy
N
Tikhmyanova
Nadia
N
Coleman
Thomas R
TR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism* / Q000502:physiology*; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014981:Xenopus; D029867:Xenopus Proteins
C103706:CDC6 protein, human; C118388:Cdc6 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D009687:Nuclear Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases