The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides

Replication licensing factor (RLF) is involved in preventing re-replication of chromosomal DNA in a single cell cycle, and previously has been separated into two components termed RLF-M and RLF-B. Here we show that Xenopus RLF-M consists of all six members of the MCM/P1 protein family, XMcm2-XMcm7. The six MCM/P1 polypeptides co-eluted on glycerol gradients and gel filtration as complexes with a mol. wt of approximately 400 kDa. In crude Xenopus extract, all six MCM/P1 polypeptides co-precipitated with anti-XMcm3 antibody, although only XMcm5 quantitatively co-precipitated from purified RLF-M. Further fractionation separated RLF-M into two sub-components, one consisting of XMcms 3 and 5, the other consisting of XMcms 2, 4, 6 and 7. Neither of the sub-components provided RLF-M activity. Finally, we show that all six MCM/P1 proteins bind synchronously to chromatin before the onset of S-phase and are displaced as S-phase proceeds. These results strongly suggest that complexes containing all six MCM/P1 proteins are necessary for replication licensing.