Identification and characterization of Saccharomyces cerevisiae Cdc6 DNA-binding properties

Recent studies have shown that Cdc6 is an essential regulator in the format ion of DNA replication complexes. However, the biochemical nature of the Cd c6 molecule is still largely unknown. In this report, we present evidence t hat the Saccharomyces cerevisiae Cdc6 protein is a double-stranded DNA-bind ing protein. First, we have demonstrated that die purified yeast Cdc6 can b ind to double-stranded DNA (dissociation constant similar to 1 x 10(-7) M), not to single-stranded DNA, and that the Cdc6 molecule is a homodimer in i ts native form. Second, we show that GST-Cdc6 fusion proteins expressed in Escherichia coli bind DNA in an electrophoretic mobility shift assay. Cdc6 antibodies and GST antibodies, but not preimmune serum, induce supershifts of GST-Cdc6 and DNA complexes in these assays, which also showed that GST-C dc6 binds to various DNA probes without apparent sequence specificity. Thir d, the minimal requirement for the binding of Cdc6 to DNA has been mapped w ithin its N-terminal 47-amino acid sequence (the NP6 region). This minimal binding domain shows identical DNA-binding properties to those possessed by full-length Cdc6. Fourth, the CST-NP6 protein competes for DNA binding wit h distamycin A, an antibiotic that chelates DNA within the minor groove of the A+T-rich region. Finally, site-direct mutagenesis studies revealed that the (KRKK)-K-29 region of Cdc6 is essential for Cdc6 DNA-binding activity. To further elucidate the function of Cdc6 DNA binding in vivo, we demonstr ated that a binding mutant of Cdc6 fails to complement either cdc6-1 temper ature-sensitive mutant cells or Delta cdc6 null mutant cells at the nonperm issive temperature. The mutant gene also conferred growth impairments and i ncreased the plasmid loss in its host, indicative of defects in DNA synthes is. Because the mutant defective in DNA binding also fails to stimulate Abf 1 ARS1 DNA-binding activity, our results suggest that Cdc6 DNA-binding acti vity may play a pivotal role in the initiation of DNA replication.