We used native mass spectrometry (MS) to investigate how the architecture of the p21 DNA response element (DNA-RE) controls the binding mode of the tumor suppressor protein p53. We analyzed tetrameric full-length wild-type p53 and compared its DNA binding behavior with a dimeric variant, p53L344A. In total, 37 DNA constructs derived from p21 DNA-RE were examined via native MS, including full-site sequences, isolated half-sites, variants differing in length and composition, and random DNA sequences. The aim was to define the minimal DNA requirements for p53 binding using native MS as a robust platform for comparative analysis of p53:DNA assemblies. Our results show that flanking regions of the full 20-bp DNA-RE have no influence on the initial formation of p53:DNA complexes. However, the complete 20-bp site is required for both p53wild-type and p53L344A to bind to DNA as tetramers. Binding of a single dimer to an isolated half-site is insufficient for generating the stable tetrameric p53:DNA complex. These findings indicate that dimer-dimer interactions are crucial for stabilizing the tetrameric p53:DNA complex.
Keywords: 20-bp; DNA; DNA-RE; MS; binding; isolated; native; p21; p53; p53:DNA; tetrameric.