THE POSSIBLE INVOLVEMENT OF REPLICATION-RELATED PROTEINS WITH A DEAD-BOX-LIKE MOTIF IN CELL-FREE DNA-REPLICATION OF XENOPUS EGGS

Two types of antibodies were prepared: one directed against an oligopeptide specific to P1 protein, a mammalian homologue of yeast MCM3, and the other against an oligopeptide with a DEAD box motif, which is a highly conserved sequence in the P1 protein family. Immunoprecipitation of the eluate from anti-P1 family IgG-bound beads, which had been incubated in Xenopus egg extracts, with anti-P1 IgG-bound beads revealed that three proteins were coprecipitated. Two proteins remained in the supernatant after the immunoprecipitation of the eluate from anti-P1 family IgG-bound beads with anti-P1 IgG-bound beads. The immunodepleted extracts with anti-P1 family IgG-bound beads showed much lower DNA replication activity than did mock-treated extracts. Recovery of replication was achieved by supplementing the depleted extracts with both the eluate from anti-P1 IgG-bound beads and the supernatant obtained after the immunoprecipitation of the eluate with anti-P1 IgG-bound beads but not by supplementing the extracts with only the proteins eluted from anti-P1 IgG-bound beads. These findings suggest that some proteins containing a DEAD-box-like motif as well as mammalian homologues of yeast MCM2, MCM3 and CDC46 play an important role in cell-free DNA replication of Xenopus eggs. (C) 1995 Academic Press,Inc.