Related Experiment Videos
Anti-oncogene product p53 binds DNA helicase
T Sakurai1, M Suzuki, T Sawazaki
1Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Japan.
Abstract:
An oncogene product, p53, interacts with a simian virus 40-encoded T-antigen, which is an initiation protein for the viral DNA replication and also works as DNA helicase during elongation. Here we examine the interaction of p53 with cellular DNA helicase. A recombinant human wild type p53 fused with glutathione S-transferase was immobilized on glutathione-agarose as a ligand for affinity column. Hela cell extract was applied to the p53 column and the adsorbed proteins were eluted with buffers containing salt, 50% ethylene glycol, and glutathione. The ethylene glycol fraction contained a number of p53 binding proteins, and this fraction showed a DNA helicase activity measured by the displacement of DNA fragment from partially duplexed M13 DNA. The DNA helicase translocated in a 5'-to-3' direction on the single-stranded DNA using ATP as an energy source. The glutathione fraction that contained the p53 glutathione S-transferase fused protein also showed the same activity. The corresponding fractions from a control column carrying glutathione S-transferase showed only a trace amount of activity of DNA helicase. Therefore, the binding may be specific. Furthermore, an anti-p53 antibody column retained a p53-DNA helicase complex when the crude extracts of human placenta and of osteosarcoma cells were applied. These results indicate that p53 physically interacts with DNA helicase in vitro as well as in vivo.
Insights
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.