Measuring the formaldehyde Protein-DNA cross-link reversal rate

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.

Figure 1

Formaldehyde protein–DNA cross-link/reversal mechanism between Lys and dG. All steps in the reaction mechanism are reversible. Lysine-containing proteins (R₁) initially attack the electrophilic carbon of formaldehyde. After loss of water, the resultant Schiff base is susceptible to nucleophilic attack by the exocyclic amine of deoxyguanosine residues (R₂) to form an aminal linkage between protein (R₁) and DNA (R₂) components. Both the cross-linking and cross-link reversal steps are demarcated by the rectangle.

Figure 2

Temperature dependence of the cross-link reversal rate. The percentage of protein-free DNA measured by qPCR is plotted versus time for samples at four different temperatures: 4, 23, 37, and 47 °C (A). Each point is the average qPCR signal for the three different genes INO1, X-element, and 25S rDNA. The error bars are the standard deviation of qPCR signals. A best fit linear regression is shown for each temperature trace. The slope of each line was determined and plotted in panel B as a function of temperature (°C). These values represent the reverse cross-linking rate. The rate of reverse cross-linking as a function of temperature was fit to an exponential.