Related Experiment Videos
Phosphorylation of HeLa cell multiprotein DNA polymerase alpha complex: impact on activity and partial purification
1Department of Biochemistry, University of Nebraska Medical Center, Omaha 68105-1065.
Abstract:
Phosphorylation is a major post-translational regulatory mechanism and plays a key role in transduction of mitogenic signals in cell proliferation. The role of phosphorylation and dephosphorylation in regulating the activities of a multiprotein DNA polymerase alpha complex was examined. Treatment of the HeLa cell multiprotein DNA polymerase alpha with calf intestinal alkaline phosphatase resulted in the inactivation of DNA polymerase alpha and DNA primase but had no effect on deoxyribonuclease- and primer-recognition proteins. A protein kinase co-purified with the multiprotein DNA polymerase alpha and was partially purified from HeLa cells. The partially purified kinase was active in phosphorylating dephosphorylated polymerase alpha and used casein and histones as exogenous substrates. This study demonstrates that phosphorylation-dephosphorylation may have modulated the activities of DNA replicative enzymes and suggests a role for specific phosphatases and kinases in this process.
Insights
Phosphorylation and dephosphorylation regulate DNA polymerase alpha complex activity. Specific kinases and phosphatases modulate DNA replicative enzymes involved in cell proliferation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Phosphorylation is a key post-translational modification regulating cell signaling pathways.
- Mitogenic signals crucial for cell proliferation are transduced via phosphorylation cascades.
- The regulation of DNA polymerase alpha complex activity is essential for DNA replication.
Purpose of the Study:
- To investigate the role of phosphorylation and dephosphorylation in modulating the activity of the multiprotein DNA polymerase alpha complex.
- To identify the involvement of specific protein kinases and phosphatases in regulating DNA replicative enzymes.
Main Methods:
- Treatment of HeLa cell multiprotein DNA polymerase alpha complex with calf intestinal alkaline phosphatase.
- Partial purification of a co-purifying protein kinase from HeLa cells.
- Assay of kinase activity using dephosphorylated polymerase alpha, casein, and histones as substrates.
Main Results:
- Calf intestinal alkaline phosphatase inactivated DNA polymerase alpha and DNA primase activities.
- Deoxyribonuclease and primer-recognition protein activities remained unaffected by phosphatase treatment.
- A partially purified protein kinase phosphorylated dephosphorylated polymerase alpha and acted on exogenous substrates like casein and histones.
Conclusions:
- Phosphorylation and dephosphorylation are critical regulatory mechanisms for DNA replicative enzymes.
- Specific phosphatases and kinases play a significant role in modulating the activity of the DNA polymerase alpha complex.
- These regulatory processes are likely involved in controlling cell proliferation through DNA replication.