Role of the second-largest subunit of DNA polymerase alpha in the interaction between the catalytic subunit and hyperphosphorylated retinoblastoma protein in late S phase.
DNA polymerase alpha (pol-alpha) 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-alpha are phosphorylated by Cdk-cyclin complexes, with p68 being hyperphosphorylated by cyclin-dependent kinases in G(2) phase of the cell cycle. The activity of Cdk2-cyclin A increases during late S phase and peaks in G(2) phase. We have now examined the role of p68 in the interaction between the catalytic subunit of pol-alpha and hyperphosphorylated retinoblastoma protein (ppRb) and in the stimulation of the polymerase activity of pol-alpha by ppRb. With the use of recombinant proteins, we found that nonphosphorylated p68 inhibited the stimulation of pol-alpha 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-alpha which may result in a conformational change in pol-alpha facilitating its interaction with and activation by ppRb.
Takemura
Masaharu
M
Department of Biology, Faculty of Science, Tokyo University of Science, RIKADAI, Kagurazaka 1-3, Tokyo 162-8601, Japan. takemura@rs.kagu.tus.ac.jp
Yoshida
Shonen
S
Akiyama
Tetsu
T
Kitagawa
Masatoshi
M
Yamada
Yoshiji
Y
D000818:Animals; D019925:Cyclin A / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004256:DNA Polymerase I / Q000502:physiology*; D006367:HeLa Cells; D006570:Heterochromatin / Q000378:metabolism; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D021122:Protein Subunits / Q000502:physiology*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D016196:S Phase / Q000502:physiology*
D019925:Cyclin A; D006570:Heterochromatin; D021122:Protein Subunits; D016160:Retinoblastoma Protein; D051357:Cyclin-Dependent Kinase 2; D004256:DNA Polymerase I