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Figure 4A, D and Figure S4 led to the conclusion that CAF-1 associates with multimers of H3/H4 in the absence of DNA. We now believe that the mixed fluorophore FRET… Click to show full abstract
Figure 4A, D and Figure S4 led to the conclusion that CAF-1 associates with multimers of H3/H4 in the absence of DNA. We now believe that the mixed fluorophore FRET assay interpreted to form this conclusion is not reliable for proving H3/H4 tetramerization by CAF-1. Despite the reproducibility of this assay and the concordance of the control experiments in Figures 4B, 4C, and S3 with the established literature, the assay appears to give an erroneous FRET effect for the CAF1-H3/H4 interaction for unknown reason(s). This fluorescence assay can be used to report on binding affinities and gives reliable KD values for H3/H4 with CAF-1, but it does not report the proximity between H3/H4 dimers. We suspect that the spectral changes that occur with CAF-1 are a result of fluorophore quenching and/or weak photostability. As such, we caution against the use of this approach in Figure 4A to study histone oligomerization.
Journal Title: Nucleic Acids Research
Year Published: 2017
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