Protein interaction and cellular localization of human CDC45.
CDC45, which plays a role in eukaryotic DNA replication, is a member of the CMG (CDC45/MCM2-7/GINS) complex that is thought to function as a replicative DNA helicase. However, the biochemical properties of CDC45 are not fully understood. We systematically examined the interactions of human CDC45 with MCM2-7, GINS and other replication proteins by immunoprecipitation. We found that CDC45 can directly interact with all MCM2-7 proteins; with PSF2, PSF3 and SLD5 in GINS subunits; and with replication protein A2 (RPA2), AND-1 and topoisomerase 2-binding protein 1. These results are consistent with the notion that CDC45 plays a role in progression of DNA replication forks. Experiments using antibodies against CDC45 show that the level of CDC45 recovered from the Triton-insoluble chromatin-containing fraction is peaked at middle of S phase in synchronized HeLa cells. However, incubation of the Triton-insoluble fraction with nucleases resulted in recovery of less than half the amount of CDC45 in the nuclease-sensitive fraction; this result is in contrast with RPA1 and proliferating cell nuclear antigen distribution. These results indicate that a considerable portion of CDC45 localizes in a region other than the DNA replication forks in nuclei or it localizes on the replication forks but it is not fractionated with the fork proteins owing to its tight association with presumably nuclear scaffolds.
College of Science, Ibaraki University, Mito, Ibaraki 351-8511, Japan.
Junichiro
J
Takaya
Shunsuke
S
Kusunoki
Yukio
Y
Ishimi
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000276:immunology / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007313:Insecta; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D060066:Protein Interaction Maps; D051716:Replication Protein A / Q000378:metabolism; D016196:S Phase
D000071181:ATP Binding Cassette Transporter, Subfamily B, Member 2; D018528:ATP-Binding Cassette Transporters; C113571:CDC45 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C556406:GINS2 protein, human; C521091:GINS4 protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; C073119:TAP1 protein, human; C110513:TOPBP1 protein, human; C544202:WDHD1 protein, human; C516794:RPA2 protein, human; C084859:MCM2 protein, human; C101624:MCM7 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064168:Minichromosome Maintenance Complex Component 7