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Anti-oncogene product p53 binds DNA helicase
T Sakurai1, M Suzuki, T Sawazaki
1Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Japan.
Experimental Cell Research
|November 1, 1994
Summary
The tumor suppressor protein p53 interacts with cellular DNA helicase. This interaction was confirmed using affinity chromatography and antibody-based assays, suggesting a role for p53 in DNA replication and repair.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- The p53 protein is a tumor suppressor involved in cellular responses to DNA damage.
- Simian virus 40 T-antigen, a viral protein, interacts with p53 and possesses DNA helicase activity.
- The interaction of p53 with cellular DNA helicases is not well understood.
Purpose of the Study:
- To investigate the physical interaction between the p53 protein and cellular DNA helicases.
- To determine if p53 binds to DNA helicase with specificity.
- To explore the potential role of p53 in DNA replication or repair processes involving helicases.
Main Methods:
- Affinity chromatography using recombinant human wild-type p53 fused to glutathione S-transferase (GST) immobilized on a glutathione-agarose column.
- Elution of bound proteins from the p53 affinity column using buffers with varying salt concentrations, ethylene glycol, and glutathione.
- Assay of DNA helicase activity using partially duplexed M13 DNA to measure the displacement of DNA fragments.
- Immunoaffinity purification using an anti-p53 antibody column with crude extracts from human placenta and osteosarcoma cells.
Main Results:
- A fraction eluted from the p53 affinity column exhibited significant DNA helicase activity, indicating p53 binds to a DNA helicase.
- The DNA helicase activity observed was ATP-dependent and translocated in a 5'-to-3' direction on single-stranded DNA.
- Control experiments using a GST-only column showed minimal helicase activity, suggesting specific binding of p53 to the helicase.
- An anti-p53 antibody column successfully retained a p53-DNA helicase complex from cellular extracts, confirming the interaction in vitro and in vivo.
Conclusions:
- The tumor suppressor protein p53 physically interacts with cellular DNA helicase.
- This interaction is specific and occurs in both in vitro and in vivo settings.
- The findings suggest a potential role for p53 in regulating DNA helicase function during DNA replication or repair.