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Interaction of p53 and DNA-PK in response to nucleoside analogues: potential role as a sensor complex for DNA damage
G Achanta1, H Pelicano, L Feng
1Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Abstract:
Therapeutic nucleoside analogues such as ara-C, gemcitabine, and fludarabine exert their cytotoxic activity against cancer cells mainly by incorporation into DNA and disruption of further DNA synthesis, resulting in the triggering of apoptosis. However, the molecules that recognize the incorporated analogues in DNA and subsequently initiate the downstream cellular responses remain to be identified. Here, we report that the DNA-dependent protein kinase (DNA-PK) and p53 are able to form a protein complex that interacts with the gemcitabine-containing DNA and plays a role in signaling to apoptotic pathways. DNA-PK/Ku and p53 were copurified in a protein fraction that binds to gemcitabine-containing DNA in preference to normal DNA. Immunoprecipitation experiments revealed that the two proteins physically associate in a complex. Treatment with gemcitabine resulted in an increase of DNA-PK and p53 protein and an increase in the phosphorylation of p53 at Ser15. Furthermore, confocal microscopy demonstrated a colocalization of DNA-PK and p53 to the nucleus in cells treated with gemcitabine. The nuclear localization of the DNA-PK/p53 complex was coincident with the induction of apoptosis in these cells. Although the wild-type p53 present in the protein complex exhibited 3'-5' exonuclease activity, it was incapable of excising the incorporated gemcitabine from DNA. The binding of the p53/DNA-PK complex to DNA substantially blocked further DNA synthesis by DNA polymerases alpha and epsilon in vitro, indicating a stalling of this complex at the site of drug incorporation. These data suggest that DNA-PK and p53 may form a sensor complex that detects the disruption of DNA replication caused by nucleoside analogue incorporation and may subsequently signal for apoptosis.
Insights
DNA-dependent protein kinase (DNA-PK) and p53 form a complex that detects gemcitabine in DNA, signaling cancer cell apoptosis. This sensor complex stalls DNA synthesis, initiating programmed cell death pathways.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Therapeutic nucleoside analogues like gemcitabine induce cancer cell death by incorporating into DNA and inhibiting synthesis.
- The specific molecular mechanisms recognizing these DNA-incorporated analogues and initiating apoptosis remain largely unknown.
Purpose of the Study:
- To identify the proteins that recognize gemcitabine-incorporated DNA and mediate downstream apoptotic signaling.
- To elucidate the role of DNA-dependent protein kinase (DNA-PK) and p53 in the cellular response to gemcitabine.
Main Methods:
- Copurification of DNA-PK/Ku and p53 with gemcitabine-containing DNA.
- Immunoprecipitation to confirm physical association between DNA-PK and p53.
- Confocal microscopy to assess protein localization and Western blotting for protein levels and phosphorylation.
Main Results:
- A protein complex of DNA-PK and p53 was identified, preferentially binding to gemcitabine-containing DNA.
- Gemcitabine treatment increased DNA-PK and p53 levels and p53 phosphorylation at Ser15.
- The DNA-PK/p53 complex localized to the nucleus, coinciding with apoptosis induction and blocking DNA synthesis in vitro.
Conclusions:
- DNA-PK and p53 form a novel sensor complex that recognizes DNA damage induced by gemcitabine incorporation.
- This complex plays a critical role in signaling apoptotic pathways by stalling DNA replication.
- The findings provide new insights into the mechanism of action of nucleoside analogues in cancer therapy.