Identification of proteins that may directly interact with human RPA.
RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.
Nakaya
Ryou
R
College of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 351-8511, Japan.
Takaya
Junichiro
J
Onuki
Takeshi
T
Moritani
Mariko
M
Nozaki
Naohito
N
Ishimi
Yukio
Y
D018797:Cell Cycle Proteins / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C066470:MCM3 protein, human; C489423:MCM5 protein, human; D009687:Nuclear Proteins; D051716:Replication Protein A; C516794:RPA2 protein, human; C084859:MCM2 protein, human; C099243:MCM4 protein, human; C103258:MCM6 protein, human; C101624:MCM7 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D064126:Minichromosome Maintenance Complex Component 3; D064148:Minichromosome Maintenance Complex Component 4; D064151:Minichromosome Maintenance Complex Component 6; D064168:Minichromosome Maintenance Complex Component 7