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... More
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.
