Sequence-Dependent Local Environment in P53 Response Element Probed by Site-Directed Spin Labeling

Abstract

The p53 response elements (RE) are a family of DNAs that are specifically recognized by the tumor suppressor protein p53. Binding of p53 to REs leads to transcription activation or suppression of a diverse set of genes that regulate a number of key biological processes. The molecular basis of p53-RE interaction has been a topic of intensive studies, however, it remains unclear how this one p53 protein can specifically recognize a large number of DNA targets in order to elicit diverse functions. As sequence dependent properties of DNAs are a key determinant in protein-DNA interaction, information on local structure and dynamic features in p53 RE is important in deciphering p53-RE recognition. Towards this goal, we report results on probing p53-RE using site-directed spin labeling, where a site-specifically attached nitroxide radical is monitored to obtain structural and dynamic information of the parent macromolecule. A nucleotide-independent labeling scheme is used to scan the nitroxide along RE sequences, and continuous-wave electron paramagnetic resonance spectroscopy is used to monitor the rotational motion of the nitroxide, which encodes DNA structural and dynamic properties at the labeling site. Spectral variations have been observed, with analyses of which reveals sequence-specific local environment within the RE. This will aid in understanding p53 recognition of target DNAs.