Direct interaction of p21 with p50, the small subunit of human DNA polymerase delta.
Using a yeast two-hybrid screening technique and the p50 subunit of human DNA polymerase delta (pol delta) as a bait, p21 was found to interact with the p50 subunit of pol delta. A direct interaction between p21 and p50 was confirmed by using ELISA and pull-down assays with purified proteins. The interaction sites between p50 and p21 were mapped by pull down assays with GST deletion mutants. Residues 127-193 constitute the primary interaction region on p50 to which p21 binds, while p50 binds to the C-terminal 26 residues of p21. A histone kinase activity was associated with the highly purified calf thymus pol delta and addition of purified recombinant p21 inhibited the kinase activity in a dose dependent manner. p50 is phosphorylated in vivo and can be phosphorylated by CDK2/cyclinA in vitro. In vivo evidence of p21 association with p50 was obtained by coimmunoprecipitation using MCF7 cells. It was also shown that the association of p21 with p50 and other components of the pol delta complex increased in MCF7 cells treated with adriamycin. Our results suggested that p50 might target or anchor p21 to pol delta complex upon certain DNA damage such as adriamycin treatment.
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
Hao
H
Li
Bin
B
Xie
Amal
A
Rahmeh
Yajing
Y
Zhou
Marietta Y W T
MY
Lee
D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D004258:DNA Polymerase III / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004317:Doxorubicin / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D006656:Protamine Kinase / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D025941:Protein Interaction Mapping; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D010750:Phosphoproteins; D021122:Protein Subunits; D004317:Doxorubicin; D006656:Protamine Kinase; D051357:Cyclin-Dependent Kinase 2; D004258:DNA Polymerase III