Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity

Replication licensing factor (RLF) is a multiprotein complex involved in en suring that chromosomal DNA replicates only once in a single cell cycle. It comprises two components, termed RLF-M and RLF-B. Purified RLF-M consists of a mixture of complexes containing all six members of the MCM/P1 family o f minichromosome maintenance proteins. The precise composition of these dif ferent complexes and their contribution to RLF-M activity has been unclear. Here we show that in Xenopus extracts, MCM/P1 proteins mainly form heteroh examers containing each of the six proteins. This heterohexamer is readily split into subcomplexes, whose interactions and subunit composition we char acterize in detail. We show for the first time an ordered multistep assembl y pathway by which the heterohexamer can be reformed from the subcomplexes. Importantly, this novel pathway is essential for DNA replication, since on ly the full heterohexamer can bind productively to chromatin and provide RL F-M activity.