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.
Xenopus laevis early development is characterized by rapid and synchronous cleavage cycles, which consist of alternating S and M phases. At midblastula transition, zygotic transcription begins and these cleavage cycles are replaced by longer cell division cycles that include gap phases and checkpoints. Herein, we demonstrate developmentally regulated Cdc6 isoform switching that contributes to this developmental cell cycle remodeling. Cdc6 is an essential component of the eukaryotic DNA replication machine that licenses each origin to one round of DNA replication each cell division cycle. The originally characterized Xenopus Cdc6 isoform (here termed Xcdc6A) and a novel isoform (Xcdc6B) have divergent N-terminal regulatory regions and different temporal patterns of expression. Although abundant in the early embryo, Xcdc6A becomes undetectable following midblastula transition. In contrast, while Xcdc6B is present in the early embryo, it is nonfunctional, as judged by lack of chromatin binding. In somatic tissue, however, Xcdc6B binds chromatin and its inhibition blocks entry into S phase. This is the first example of developmental regulation of Cdc6, raising intriguing implications for cell cycle remodeling during embryogenesis.
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