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Measuring the Formaldehyde Protein–DNA Cross-Link Reversal Rate

J. Kennedy‐Darling,Lloyd M. Smith

Published 2014 in Analytical Chemistry

ABSTRACT

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.

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