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Proteolytic cleavage of p53 mutants in response to mismatched DNA
T Mee1, A L Okorokov, S Metcalfe
1YCR P53 Research Group, Department of Biology, University of York, UK.
Abstract:
Interaction of p53 with mismatched DNA induces proteolytic cleavage with release of a 35-kDa protein fragment from the p53-DNA complexes. The 35-kDa cleavage product is activated for specific biochemical function(s) and may play a role in the cellular response to DNA damage (Molinari et al (1996) Oncogene 13: 2077-2086; Okorokov et al (1997) EMBO J 16: 6008-6017). In the present study we have asked if mutants of p53 retain the ability to undergo similar proteolytic cleavage, and compared sequence-specific 'DNA contact' with 'structural' mutants commonly found in human cancer. In addition, a series of phosphorylation site mutants were generated to investigate the possible effects of phosphorylation/dephosphorylation on the proteolytic cleavage of p53. All mutants tested bound to a mismatched DNA target in vitro. Moreover, studies in vitro and in vivo indicate that p53 mutants with intact conformational structure (as determined by immunoreactivity with PAb246 and PAb1620) retain the ability to undergo proteolytic cleavage similar, if not identical, to the wild-type p53 protein. Our results suggest that the capacity for p53 to bind mismatched DNA is independent of structural conformation of the central core domain. Proteolytic cleavage, however, is crucially dependent upon a wild-type conformation of the protein.
Insights
Wild-type p53 protein undergoes proteolytic cleavage when interacting with mismatched DNA. Mutants with intact structure retain this ability, but cleavage requires a wild-type protein conformation.
Area of Science:
- Molecular Biology
- Cancer Research
- Protein Biochemistry
Background:
- p53 protein interacts with mismatched DNA, leading to proteolytic cleavage and release of a 35-kDa fragment.
- This fragment may play a role in cellular DNA damage response.
- Previous studies have established the cleavage mechanism for wild-type p53.
Purpose of the Study:
- To investigate if p53 mutants retain the ability for proteolytic cleavage upon interaction with mismatched DNA.
- To compare sequence-specific DNA contact mutants with structural mutants found in human cancer.
- To explore the effect of phosphorylation/dephosphorylation on p53 proteolytic cleavage.
Main Methods:
- In vitro and in vivo studies were conducted.
- Mutants of p53 were generated, including sequence-specific, structural, and phosphorylation site mutants.
- Immunoreactivity with PAb246 and PAb1620 antibodies was used to assess conformational structure.
Main Results:
- All tested p53 mutants successfully bound to mismatched DNA targets in vitro.
- p53 mutants with intact conformational structure demonstrated proteolytic cleavage similar to wild-type p53.
- The ability to bind mismatched DNA was independent of the central core domain's structural conformation.
Conclusions:
- Proteolytic cleavage of p53 is critically dependent on a wild-type protein conformation.
- The capacity of p53 to bind mismatched DNA is not dependent on its structural conformation.
- These findings shed light on the structural requirements for p53 function in DNA damage response.