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Identification of genomic DNA sequences bound by mutant p53 protein (Gly245-->Ser) in vivo
1Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany.
Abstract:
Mutant p53 proteins were shown to exert complex DNA-interactions in vitro, like binding to MAR-DNA, but so far it is unknown whether such interactions also occur in vivo. Therefore we analysed the binding of mutant (mut) p53 (Gly245-->Ser) in Onda 11 glioma cells to cellular DNA in vivo, using p53-specific chromatin immunoprecipitation (CHIP) after in vivo cross-linking of mut p53 to genomic DNA with cisplatin. We identified genomic DNA fragments to which mut p53 (Gly245-->Ser) could be cross-linked in vivo. Purified recombinant mut p53 (Gly245-->Ser) was able to bind specifically to such elements in PCR-EMSA in vitro, supporting the idea that this mut p53 protein interacts with genomic DNA in vivo. The genomic DNA fragments identified are vastly different in sequence, but display as a common feature a high likelihood to adopt a non B-DNA conformation. Therefore we propose that structural determinants within these DNA elements are important for their interaction with mut p53 (Gly245-->Ser) in vivo. Oncogene (2000) 19, 4178 - 4183
Insights
Mutant p53 proteins bind to specific DNA regions in glioma cells in vivo. This interaction is mediated by DNA structural features, not sequence, suggesting a novel mechanism for mutant p53 function.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Mutant p53 proteins exhibit complex DNA-binding in vitro.
- In vivo DNA interactions of mutant p53 remain largely uncharacterized.
Purpose of the Study:
- To investigate in vivo DNA binding of mutant p53 (Gly245-->Ser) in Onda 11 glioma cells.
- To identify genomic DNA fragments interacting with mutant p53 in vivo.
Main Methods:
- Chromatin immunoprecipitation (CHIP) with p53-specific antibodies.
- In vivo cross-linking of mutant p53 to genomic DNA using cisplatin.
- Polymerase chain reaction-electrophoretic mobility shift assay (PCR-EMSA) for in vitro binding validation.
Main Results:
- Identified specific genomic DNA fragments cross-linked to mutant p53 (Gly245-->Ser) in vivo.
- Recombinant mutant p53 (Gly245-->Ser) specifically bound to these identified DNA elements in vitro.
- Interacting DNA fragments, despite sequence diversity, share a high propensity for non-B DNA conformations.
Conclusions:
- Mutant p53 (Gly245-->Ser) interacts with genomic DNA in vivo.
- The interaction is likely driven by DNA structural determinants, such as non-B DNA conformations, rather than specific sequences.
- This suggests a novel mechanism for mutant p53's role in cellular processes.