DNA damage activates p53 through a phosphorylation–acetylation cascade. p53 is a tumor suppressor that responds to DNA damage by stabilizing and activating transcription. Post-translational modifications, including phosphorylation and acetylation, regulate p53's stability and DNA-binding activity. This study shows that p53 is acetylated by two histone acetyltransferases (HATs), p300 and PCAF, at distinct sites. p300 acetylates Lys-382 in the carboxy-terminal region, while PCAF acetylates Lys-320 in the nuclear localization signal. Acetylation at these sites enhances p53's DNA-binding ability. In vivo, exposure to UV or ionizing radiation leads to acetylation of Lys-382 and phosphorylation of Ser-33 and Ser-37. In vitro, phosphorylated amino-terminal p53 peptides inhibit acetylation by HATs. These findings suggest that DNA damage enhances p53 activity through carboxy-terminal acetylation, which is directed by amino-terminal phosphorylation. The study also shows that phosphorylation of p53 at Ser-37 and Ser-33 enhances its interaction with p300 and PCAF, promoting acetylation. Acetylation of p53 at Lys-382 and Lys-320 is detected in response to DNA damage, with acetylation at Lys-382 being more prominent. Phosphorylation of p53 at Ser-37 is also observed in response to DNA damage. These results indicate that p53 is acetylated in vivo in response to DNA damage, with acetylation at Lys-382 and Lys-320 being specific to different damage types. The study highlights the role of phosphorylation in modulating acetylation, which in turn enhances p53's ability to bind DNA specifically. The findings suggest that DNA damage activates p53 through a cascade of post-translational modifications, including phosphorylation and acetylation, which are critical for p53's function as a transcription factor.DNA damage activates p53 through a phosphorylation–acetylation cascade. p53 is a tumor suppressor that responds to DNA damage by stabilizing and activating transcription. Post-translational modifications, including phosphorylation and acetylation, regulate p53's stability and DNA-binding activity. This study shows that p53 is acetylated by two histone acetyltransferases (HATs), p300 and PCAF, at distinct sites. p300 acetylates Lys-382 in the carboxy-terminal region, while PCAF acetylates Lys-320 in the nuclear localization signal. Acetylation at these sites enhances p53's DNA-binding ability. In vivo, exposure to UV or ionizing radiation leads to acetylation of Lys-382 and phosphorylation of Ser-33 and Ser-37. In vitro, phosphorylated amino-terminal p53 peptides inhibit acetylation by HATs. These findings suggest that DNA damage enhances p53 activity through carboxy-terminal acetylation, which is directed by amino-terminal phosphorylation. The study also shows that phosphorylation of p53 at Ser-37 and Ser-33 enhances its interaction with p300 and PCAF, promoting acetylation. Acetylation of p53 at Lys-382 and Lys-320 is detected in response to DNA damage, with acetylation at Lys-382 being more prominent. Phosphorylation of p53 at Ser-37 is also observed in response to DNA damage. These results indicate that p53 is acetylated in vivo in response to DNA damage, with acetylation at Lys-382 and Lys-320 being specific to different damage types. The study highlights the role of phosphorylation in modulating acetylation, which in turn enhances p53's ability to bind DNA specifically. The findings suggest that DNA damage activates p53 through a cascade of post-translational modifications, including phosphorylation and acetylation, which are critical for p53's function as a transcription factor.