Protocol

Site-Specific Protein-DNA Photo-Cross-Linking: Analysis of Structural Organization of Protein-DNA and Multiprotein-DNA Complexes

This protocol was adapted from "Characterization of Protein Complexes," Chapter 10, in Proteins and Proteomics (ed. Simpson). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

INTRODUCTION

Site-specific protein-DNA photo-cross-linking is able to define positions of proteins relative to DNA within large multiprotein-DNA complexes. Chemical and enzymatic reactions are used to prepare a DNA fragment containing a phenyl-azide photoactivatible cross-linking agent and an adjacent radiolabel incorporated at a single, defined DNA phosphate. The multiprotein-DNA complex of interest is formed using the site-specifically derivatized DNA fragment. The complex is then UV-irradiated, initiating covalent cross-linking with proteins in direct physical proximity to the cross-linking agent. Extensive nuclease digestion is performed to eliminate uncross-linked DNA and convert cross-linked DNA to a cross-linked, radiolabeled nucleotide "tag." The "tagged" proteins are identified, usually by denaturing polyacrylamide gel electrophoresis followed by autoradiography. The procedure is performed in a systematic fashion: At least 10 derivatized DNA fragments, each having the cross-linking agent incorporated at a defined DNA phosphate within the region of interest, are analyzed. The results of the procedure define the translational positions of proteins relative to the DNA sequence. Plotted on a three-dimensional representation of a DNA helix, the results also define the rotational orientations of proteins relative to the DNA helix axis and the groove orientations of proteins relative to the DNA major and minor grooves. Here, we present a detailed protocol for the cross-linking of protein-DNA complexes immobilized on streptavidin-coated paramagnetic beads ("on-bead" cross-linking).