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The RLF‐M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides
Author(s) -
Thömmes Pia,
Kubota Yumiko,
Takisawa Haruhiko,
Blow J.Julian
Publication year - 1997
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/16.11.3312
Subject(s) - biology , component (thermodynamics) , replication (statistics) , computational biology , genetics , virology , thermodynamics , physics
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 ∼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.