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 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.
Prokhorova
T A
TA
Cancer Research Campaign Chromosome Replication Research Group, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.
Blow
J J
JJ
D001704:Biopolymers; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D004261:DNA Replication; D004268:DNA-Binding Proteins; D064148:Minichromosome Maintenance Complex Component 4; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D009994:Osmolar Concentration; D011499:Protein Processing, Post-Translational
D001704:Biopolymers; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C099243:MCM4 protein, human; D064148:Minichromosome Maintenance Complex Component 4