Protein-protein interactions occur between p53 phosphoforms and ATM and 53BP1 at sites of exogenous DNA damage

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• The p53 protein is a crucial tumor suppressor involved in the DNA damage response.
• Phosphorylation of p53 at Serine 15 (p53(Ser15)) is a key event following DNA damage.
• p53's role in anchoring at DNA damage sites and interacting with ATM and 53BP1 requires further elucidation.

Purpose of the Study:

• To investigate the role of the p53 carboxy-terminus (C-terminus) non-specific DNA binding domain (NSDBD) in chromatin anchoring at DNA damage sites.
• To determine how the p53 C-terminus mediates interactions with ATM and 53BP1 during the DNA damage response (DDR).

Main Methods:

• Utilized exogenous YFP-p53 fusion constructs with C-terminus deletion mutants in p53-null H1299 cells.
• Employed microscopy and biochemistry to assess chromatin binding of p53 mutants pre- and post-irradiation.
• Conducted subnuclear UV-microbeam and immunoprecipitation analyses in irradiated human fibroblasts.

Main Results:

• Wild-type YFP-p53 and a C-terminal deletion mutant (YFP-p53(Δ367-393)) associated with ATM(Ser1981) and 53BP1 at chromatin after DNA damage.
• A mutant lacking the N-terminus and Ser15 (YFP-p53(Δ1-299)) bound ATM(Ser1981) but not 53BP1.
• Confirmed interactions between endogenous p53 and ATM or 53BP1 in irradiated human fibroblasts.

Conclusions:

• The p53 C-terminus, specifically its NSDBD, is critical for chromatin anchoring at DNA damage sites.
• p53 utilizes its C-terminus to facilitate interactions with ATM and 53BP1, thereby promoting the DNA damage response.
• A model is proposed where p53 rapidly responds to DNA damage by using its C-terminus for spatial facilitation of protein interactions and DDR.