Measuring the formaldehyde Protein-DNA cross-link reversal rate.
Protein-DNA binding interactions play critical roles in important cellular processes such as gene expression, cell division, and chromosomal organization. Techniques to identify and characterize these interactions often utilize formaldehyde cross-linking for stabilization of the complexes. Advantages of formaldehyde as a cross-linking reagent include cell permeability, relatively fast cross-linking kinetics, and short cross-linker length. In addition, formaldehyde cross-links are reversible, which has the advantage of allowing complexes to be dissociated if desired but may also present a problem if undesired dissociation occurs in the course of an experiment. While the kinetics of formaldehyde cross-link formation have been well-established in numerous studies, there have been no reports of the rate of cross-link dissociation, even though it is clearly a critical variable when developing a biochemical protocol involving formaldehyde cross-linking. We present here a method for measurement of the rate of formaldehyde cross-link reversal based upon the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) procedure and use it to determine the rate of cross-link reversal for cross-linked protein-DNA complexes from yeast cell lysate. The half-life of the protein-DNA cross-links varies from 179 h at 4 °C to 11.3 h at 47 °C, with a rate that increases exponentially with temperature and is independent of salt concentration.
Department of Chemistry, University of Wisconsin , 1101 University Avenue, Madison, Wisconsin 53706, United States.
Julia
J
Kennedy-Darling
Lloyd M
LM
Smith
D004247:DNA / Q000737:chemistry*; D005557:Formaldehyde / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*
D011506:Proteins; D005557:Formaldehyde; D004247:DNA