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2342625 
Journal Article 
Measuring the formaldehyde protein-- DNA cross-link reversal rate 
Kennedy-Darling, J; Smith, LM 
2014 
Yes 
Analytical Chemistry
ISSN: 0003-2700
EISSN: 1520-6882 
86 
12 
5678-5681 
English 
24848408 
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 crosslinking for stabilization of the complexes. Advantages of formaldehyde as a crosslinking reagent include cell permeability, relatively fast crosslinking kinetics, and short crosslinker length. In addition, formaldehyde crosslinks 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 crosslink formation have been well-established in numerous studies, there have been no reports of the rate of crosslink dissociation, even though it is clearly a critical variable when developing a biochemical protocol involving formaldehyde crosslinking. We present here a method for measurement of the rate of formaldehyde crosslink reversal based upon the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) procedure, and use it to determine the rate of crosslink reversal for crosslinked protein:DNA complexes from yeast cell lysate. The half-life of the protein:DNA crosslinks varies from 179 hours at 4 °C to 11.3 hours at 47 °C, with a rate that increases exponentially with temperature and is independent of salt concentration.