A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication.
During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.
Evrin
Cecile
C
DNA Replication Group, Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 0NN, United Kingdom.
Clarke
Pippa
P
Zech
Juergen
J
Lurz
Rudi
R
Sun
Jingchuan
J
Uhle
Stefan
S
Li
Huilin
H
Stillman
Bruce
B
Speck
Christian
C
D000818:Animals; D002850:Chromatography, Gel; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism* / Q000648:ultrastructure; D009841:Oligonucleotides; D011487:Protein Conformation; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014981:Xenopus; D015003:Yeasts
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D004265:DNA Helicases; C069199:MCM2 protein, S cerevisiae