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Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication
H Flores-Rozas1, Z Kelman, F B Dean
1Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Abstract:
Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.
Insights
Cdk-interacting protein 1 (Cip1) directly binds proliferating cell nuclear antigen (PCNA) to inhibit DNA synthesis. This Cip1-PCNA interaction impairs DNA polymerase delta processivity, impacting DNA replication and repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Cycle Regulation
Background:
- Cdk-interacting protein 1 (Cip1) is a p53-regulated protein inhibiting cyclin-dependent kinases (CDKs).
- Cip1 was initially found in complexes with CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA).
- PCNA acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, forming the pol delta holoenzyme.
Purpose of the Study:
- To investigate the direct inhibitory effect of Cip1 on DNA synthesis.
- To determine the mechanism by which Cip1 affects DNA replication.
- To explore the implications of the Cip1-PCNA interaction in cellular processes.
Main Methods:
- In vitro DNA synthesis assays.
- Simian virus 40 (SV40) replication assays using various polymerases.
- Surface plasmon resonance (SPR) to detect direct physical interactions.
Main Results:
- Cip1 directly inhibits DNA synthesis in vitro by binding to PCNA.
- Cip1 inhibits SV40 replication dependent on pol alpha, activator 1, PCNA, and pol delta, with inhibition overcome by excess PCNA.
- SPR confirmed a direct physical interaction between PCNA and Cip1.
- Cip1 inhibits synthesis on long DNA templates but not short ones, suggesting impaired pol delta processivity.
Conclusions:
- Cip1 directly interacts with PCNA, inhibiting DNA synthesis by impairing pol delta processivity during chain elongation.
- The Cip1-PCNA interaction has significant implications for the regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair.
- This interaction provides a novel mechanism for controlling DNA replication fidelity and cellular response to DNA damage.