TP53 is one of the most frequently mutated genes in human cancer, yet the evolutionary constraints underlying recurrent cancer hotspots remain unclear. We tested whether mammalian evolutionary conservation independently marks canonical TP53 mutation hotspots as functionally constrained.

We analysed 56 curated mammalian TP53 protein sequences using multiple sequence alignment, residue-level conservation and entropy, followed by hotspot-versus-DNA-binding-domain (DBD) background comparison and integration with PanCancer mutation recurrence. All six canonical TP53 hotspots were fully conserved across the 56 mammals (conservation = 1.000; entropy = 0.000) and were significantly more conserved than the already-conserved DBD background (Mann–Whitney p = 0.0156; permutation p = 0.0236; Cliff’s δ = 0.476). Mutation recurrence also showed a positive association with mammalian conservation (Spearman ρ = 0.430, p = 4.49 × 10⁻¹⁹), with the direction remaining robust to lineage-exclusion analyses.

These results support evolutionary constraint as a useful prioritization signal for functionally sensitive TP53 residues, while demonstrating that conservation alone does not determine cancer mutation recurrence.