Assembly of the Cdc45-Mcm2-7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins.
In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G(1)/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.
Im
Jun-Sub
JS
Department of Biology Education, Seoul National University, Seoul, 151-748, Korea.
Ki
Sang-Hee
SH
Farina
Andrea
A
Jung
Dong-Soo
DS
Hurwitz
Jerard
J
Lee
Joon-Kyu
JK
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7; D064110:Minichromosome Maintenance Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D053484:RecQ Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; C113571:CDC45 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C521092:GINS3 protein, human; D008164:Luminescent Proteins; C489613:MCM10 protein, human; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; C110513:TOPBP1 protein, human; C544202:WDHD1 protein, human; C054562:yellow fluorescent protein, Bacteria; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; C506731:RECQL4 protein, human; C084859:MCM2 protein, human; C101624:MCM7 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7; D064110:Minichromosome Maintenance Proteins; D053484:RecQ Helicases