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The RLF-M component of the replication licensing system forms complexes containing all six MCM/P1 polypeptides
P Thömmes1, Y Kubota, H Takisawa
1ICRF Clare Hall Laboratories, Potters Bar, Herts, UK.
Abstract:
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.
Insights
Replication licensing factor-M (RLF-M) requires all six MCM/P1 proteins (XMcm2-XMcm7) to prevent DNA re-replication. These proteins form a complex that binds chromatin before S-phase, indicating their necessity for replication licensing.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
- DNA Replication
Background:
- Replication licensing factor (RLF) prevents DNA re-replication within a single cell cycle.
- RLF has been previously separated into RLF-M and RLF-B components.
Purpose of the Study:
- To elucidate the composition and function of Xenopus RLF-M.
- To determine the role of MCM/P1 proteins in replication licensing.
Main Methods:
- Glycerol gradient and gel filtration to determine complex molecular weight.
- Co-immunoprecipitation using anti-XMcm3 antibody.
- Chromatin binding assays before and during S-phase.
Main Results:
- Xenopus RLF-M comprises all six MCM/P1 proteins (XMcm2-XMcm7) as a ~400 kDa complex.
- Fractionation yielded sub-components lacking RLF-M activity, highlighting the necessity of the complete complex.
- All six MCM/P1 proteins bind chromatin synchronously before S-phase and are displaced during S-phase progression.
Conclusions:
- The complete complex of six MCM/P1 proteins is essential for replication licensing in Xenopus.
- MCM/P1 protein complex assembly and chromatin binding dynamics are critical for cell cycle control of DNA replication.