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Sequential MCM/P1 subcomplex assembly is required to form a heterohexamer with replication licensing activity
1Cancer Research Campaign Chromosome Replication Research Group, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Abstract:
Replication licensing factor (RLF) is a multiprotein complex involved in ensuring 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 of minichromosome maintenance proteins. The precise composition of these different complexes and their contribution to RLF-M activity has been unclear. Here we show that in Xenopus extracts, MCM/P1 proteins mainly form heterohexamers containing each of the six proteins. This heterohexamer is readily split into subcomplexes, whose interactions and subunit composition we characterize in detail. We show for the first time an ordered multistep assembly pathway by which the heterohexamer can be reformed from the subcomplexes. Importantly, this novel pathway is essential for DNA replication, since only the full heterohexamer can bind productively to chromatin and provide RLF-M activity.
Insights
Replication licensing factor (RLF) ensures DNA replicates once per cell cycle. Researchers discovered a new assembly pathway for the MCM/P1 protein complex, crucial for DNA replication and chromatin binding.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Replication licensing factor (RLF) controls DNA replication frequency.
- RLF comprises RLF-M and RLF-B components.
- RLF-M contains MCM/P1 proteins, but their complex composition and function were unclear.
Purpose of the Study:
- To elucidate the composition and assembly of MCM/P1 protein complexes within RLF-M.
- To characterize the interactions and subunit organization of MCM/P1 subcomplexes.
- To determine the functional significance of the MCM/P1 heterohexamer assembly pathway for DNA replication.
Main Methods:
- Analysis of Xenopus egg extracts.
- Protein complex purification and characterization.
- Investigation of protein-protein interactions and subunit composition.
Main Results:
- MCM/P1 proteins primarily form heterohexamers in Xenopus extracts.
- Detailed characterization of subcomplexes formed upon heterohexamer dissociation.
- Identification of an ordered, multistep pathway for heterohexamer reformation from subcomplexes.
Conclusions:
- The full MCM/P1 heterohexamer is essential for RLF-M activity.
- Productive chromatin binding and DNA replication depend on the complete heterohexamer.
- A novel assembly pathway is critical for ensuring once-per-cell-cycle DNA replication.