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Specific and complex interactions of murine p53 with DNA
S N Weissker1, B F Müller, A Homfeld
1Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie, Universität Hamburg, Germany.
Abstract:
Biologically active mutant p53 from Balb/c mouse tumor cells (Meth A) was analysed for its specific interaction with DNA. Restricted phage lambda DNA, representing DNA of high complexity with regard to sequence and secondary structure, was used to probe for such an activity in a target-bound DNA-binding assay, using doubly immunopurified p53. A single lambda DNA fragment was specifically retained with very high affinity (KD = 10(-10) M). Specific DNA binding was shown to be an intrinsic property of p53, as it could be blocked with p53-specific monoclonal antibodies PAb122 and PAb421. The characteristics of the DNA binding of p53 to this lambda DNA fragment, as well as the structural properties of this fragment, suggested the possibility that p53 might be able to interact with nuclear matrix attachment region (MAR) DNA. Indeed, established genomic MAR elements were specifically bound by Meth A p53, whereas no binding was observed to an AT-rich control DNA. The interaction of p53 with MAR elements in vitro is compatible with the idea that p53 in vivo is involved in the regulation of replication and/or expression of cellular DNA. Complex DNA interactions were not restricted to mutant p53 from Meth A cells. Mutant p53 of a different conformational phenotype (PAb246+ 'wild-type' as opposed to PAb246- 'mutant' for p53 from Meth A cells) from minimally transformed T3T3 cells, as well as genotypic wild-type p53 expressed by a recombinant baculovirus in insect cells, exhibited similar DNA-binding properties.
Insights
Mutant p53 protein specifically binds to DNA fragments, including nuclear matrix attachment regions (MARs). This interaction, observed across different p53 forms, suggests a role in cellular DNA replication and expression regulation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The tumor suppressor protein p53 plays a critical role in maintaining genomic stability.
- Mutations in the p53 gene are common in human cancers, often leading to a "biologically active" mutant form with altered functions.
- Understanding the DNA-binding properties of mutant p53 is crucial for deciphering its role in tumorigenesis.
Purpose of the Study:
- To investigate the specific DNA-binding capabilities of biologically active mutant p53.
- To determine if mutant p53 interacts with specific DNA structures like nuclear matrix attachment regions (MARs).
- To explore whether these DNA-binding properties are conserved across different p53 conformational states and sources.
Main Methods:
- Utilized a target-bound DNA-binding assay with doubly immunopurified mutant p53 from Meth A mouse tumor cells.
- Employed restricted phage lambda DNA, a complex DNA molecule, to probe for specific interactions.
- Confirmed p53 specificity using p53-specific monoclonal antibodies (PAb122, PAb421) and tested binding to established genomic MAR elements and control DNA.
Main Results:
- Identified a specific lambda DNA fragment with very high affinity (KD = 10(-10) M) for mutant p53.
- Demonstrated that p53 specifically binds to established genomic MAR elements, but not to AT-rich control DNA.
- Observed similar DNA-binding properties in mutant p53 from T3T3 cells and genotypic wild-type p53 expressed in insect cells.
Conclusions:
- Biologically active mutant p53 exhibits specific high-affinity DNA-binding properties.
- p53 can interact with nuclear matrix attachment region (MAR) DNA, suggesting a role in DNA organization.
- These findings support the hypothesis that p53, in various forms, participates in the regulation of cellular DNA replication and/or expression.