PURIFICATION OF AN MCM-CONTAINING COMPLEXES A COMPONENT OF THE DNA-REPLICATION LICENSING SYSTEM

REPLICATION licensing factor (RLF) ensures that eukaryotic chromosomal DNA is replicated exactly once in each cell cycle(1-4). On exit from metaphase, RLF is activated and binds to or modifies chromatin. This m odification (the 'licence') is required for subsequent DNA replication ; the licence is also inactivated in the process of replication. Activ e RLF is not imported into the nucleus, so further DNA replication can not occur until the DNA is relicensed by passage throught mitosis. We have developed an assay to purify RLF from Xenopus eggs(4). Activity r esolves into two components, RLF-M and RLF-B, both of which are requir ed for licensing, RLF-M has been purified to apparent homogeneity: it consists of three polypeptides, one of which is a Xenopus homologue of the yeast MCM3 protein. Xenopus Mcm3 associates with chomatin in G1 a nd is removed during replication, consistent with its being a componen t of the RLF system.