Conformations of p53 response elements in solution deduced using site-directed spin labeling and Monte Carlo sampling

Department of Chemistry, University of Southern California, Los Angeles, CA 90089, USA and Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089, USA.
Nucleic Acids Research (Impact Factor: 9.11). 11/2013; 42(4). DOI: 10.1093/nar/gkt1219
Source: PubMed


The tumor suppressor protein p53 regulates numerous signaling pathways by specifically recognizing diverse p53 response elements (REs). Understanding the mechanisms of p53-DNA interaction requires structural information on p53 REs. However, such information is limited as a 3D structure of any RE in the unbound form is not available yet. Here, site-directed spin labeling was used to probe the solution structures of REs involved in p53 regulation of the p21 and Bax genes. Multiple nanometer distances in the p21-RE and BAX-RE, measured using a nucleotide-independent nitroxide probe and double-electron-electron-resonance spectroscopy, were used to derive molecular models of unbound REs from pools of all-atom structures generated by Monte-Carlo simulations, thus enabling analyses to reveal sequence-dependent DNA shape features of unbound REs in solution. The data revealed distinct RE conformational changes on binding to the p53 core domain, and support the hypothesis that sequence-dependent properties encoded in REs are exploited by p53 to achieve the energetically most favorable mode of deformation, consequently enhancing binding specificity. This work reveals mechanisms of p53-DNA recognition, and establishes a new experimental/computational approach for studying DNA shape in solution that has far-reaching implications for studying protein-DNA interactions.

Download full-text


Available from: Remo Rohs, Mar 19, 2014
26 Reads
  • Source
    • "The simplest substitution is a 2 0 -H (Fig. 2C), although others such as 2 0 -O-methyl and 2 0 -F, also suffice. It is worth noting that one, two, or even more phosphorothioates can be installed simultaneously within an oligonucleotide strand (Zhang et al., 2012, 2014), depending on the desired number of labels. Oligonucleotides with site-specific phosphorothioate modifications are most easily obtained via chemical synthesis using the well-established phosphoramide scheme and are available from many commercial sources. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The technique of site-directed spin labeling (SDSL) provides unique information on biomolecules by monitoring the behavior of a stable radical tag (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. In this chapter, we describe an approach in which SDSL is integrated with computational modeling to map conformations of nucleic acids. This approach builds upon a SDSL tool kit previously developed and validated, which includes three components: (i) a nucleotide-independent nitroxide probe, designated as R5, which can be efficiently attached at defined sites within arbitrary nucleic acid sequences; (ii) inter-R5 distances in the nanometer range, measured via pulsed EPR; and (iii) an efficient program, called NASNOX, that computes inter-R5 distances on given nucleic acid structures. Following a general framework of data mining, our approach uses multiple sets of measured inter-R5 distances to retrieve "correct" all-atom models from a large ensemble of models. The pool of models can be generated independently without relying on the inter-R5 distances, thus allowing a large degree of flexibility in integrating the SDSL-measured distances with a modeling approach best suited for the specific system under investigation. As such, the integrative experimental/computational approach described here represents a hybrid method for determining all-atom models based on experimentally-derived distance measurements.
    Methods in Enzymology 10/2015; 564:427-453. DOI:10.1016/bs.mie.2015.07.007 · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Sequence-dependent variation in structure and dynamics of a DNA duplex, collectively referred to as ‘DNA shape’, critically impacts interactions between DNA and proteins. Here, a method based on the technique of site-directed spin labeling was developed to experimentally map shapes of two DNA duplexes that contain response elements of the p53 tumor suppressor. An R5a nitroxide spin label, which was covalently attached at a specific phosphate group, was scanned consecutively through the DNA duplex. X-band continuous-wave electron paramagnetic resonance spectroscopy was used to monitor rotational motions of R5a, which report on DNA structure and dynamics at the labeling site. An approach based on Pearson's coefficient analysis was developed to collectively examine the degree of similarity among the ensemble of R5a spectra. The resulting Pearson's coefficients were used to generate maps representing variation of R5a mobility along the DNA duplex. The R5a mobility maps were found to correlate with maps of certain DNA helical parameters, and were capable of revealing similarity and deviation in the shape of the two closely related DNA duplexes. Collectively, the R5a probe and the Pearson's coefficient-based lineshape analysis scheme yielded a generalizable method for examining sequence-dependent DNA shapes.
    Nucleic Acids Research 08/2014; 42(18). DOI:10.1093/nar/gku695 · 9.11 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Transcription factors (TFs) influence cell fate by interpreting the regulatory DNA within a genome. TFs recognize DNA in a specific manner; the mechanisms underlying this specificity have been identified for many TFs based on 3D structures of protein-DNA complexes. More recently, structural views have been complemented with data from high-throughput in vitro and in vivo explorations of the DNA-binding preferences of many TFs. Together, these approaches have greatly expanded our understanding of TF-DNA interactions. However, the mechanisms by which TFs select in vivo binding sites and alter gene expression remain unclear. Recent work has highlighted the many variables that influence TF-DNA binding, while demonstrating that a biophysical understanding of these many factors will be central to understanding TF function.
    Trends in Biochemical Sciences 08/2014; 39(9). DOI:10.1016/j.tibs.2014.07.002 · 11.23 Impact Factor
Show more