Department of Biology, Faculty of Science, Tokyo University of Science (RIKADAI) Cancer Immunotherapy Center, Nagoya Kyoritu Hospital Department of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, University of Tokyo Department of Biochemistry 1 and the COE Program of “Medical Photonics,” Hamamatsu University School of Medicine Department of Human Functional Genomics, Life Science Research Center, Mie University
Biochimica et Biophysica Acta - Proteins and Proteomics
Retinoblastoma Rb DNA polymerase α Phosphorylation Heterochromatin Cell cycle
DNA polymerase α (pol-α) is a heterotetrameric enzyme (p180–p68–p58–p48 in mouse) that is essential for the initiation of chain elongation during DNA replication. The catalytic (p180) and p68 subunits of pol-α are phosphorylated by Cdk–cyclin complexes, with p68 being hyperphosphorylated by cyclin-dependent kinases in G2 phase of the cell cycle. The activity of Cdk2–cyclin A increases during late S phase and peaks in G2 phase. We have now examined the role of p68 in the interaction between the catalytic subunit of pol-α and hyperphosphorylated retinoblastoma protein (ppRb) and in the stimulation of the polymerase activity of pol-α by ppRb. With the use of recombinant proteins, we found that nonphosphorylated p68 inhibited the stimulation of pol-α activity by ppRb, suggesting that p68 might impede the association of ppRb with p180. Phosphorylation of p68 by Cdk2–cyclin A greatly reduced its inhibitory effect. Immunofluorescence analysis also revealed that ppRb localized at sites of DNA replication specifically in late S phase. These results suggest that Cdk–cyclin A can phosphorylate pol-α which may result in a conformational change in pol-α facilitating its interaction with and activation by ppRb.