Deformation of a P53 Response Element Revealed by Site-Directed Spin Labeling

Abstract

The tumor suppressor protein p53 is called the “guardian of the genome” and has been implicated in a growing number of biological processes, including cell cycle arrest, senescence, apoptosis, autophagy, metabolism, and aging. As a transcription factor, p53 regulates a large number of signaling pathways by interacting with a diverse set of DNAs, called p53 response elements (REs), which are composed of two decameric consensus half-sites separated by a spacer. It has been proposed that DNA conformation changes upon p53 binding, which may vary between different REs, play important roles in p53-DNA recognition. However, details on such DNA conformational changes are limited, as currently there is no high-resolution structure of unbound RE, although a number of crystal structures of tetrameric p53 fragments bound to artificial or naturally occurring REs have been reported. Here site-directed spin labeling is used to probe solution conformations of an RE regulating protein p21, a cyclin-dependent kinase inhibitor involved in cell cycle regulation. Using a nucleotide-independent nitroxide probe and Double-Electron-Electron Resonance spectroscopy, nanometer distances between various DNA sites were measured in unbound p21RE as well as in that complexed to the p53 DNA binding domain. Models of the unbound p21RE, which were selected using the measured distances from a large pool generated by Monte-Carlo simulations, reveal major conformational changes at the half-site interface as compared to the bound DNA reported in a crystal structure. These results shed light on the mechanism of DNA recognition by p53.