Cross-linking of Histone Proteins to DNA by UV Illumination of Chromatin Stained with Hoechst 33342¶

The photochemical effects of near-UV light on chromatin labeled with the vital DNA dye Hoechst 33342 (H33342) are studied. Several types of experiments demonstrate that illumination at both 365 and 410 nm results in significant cross-linking of proteins with the DNA. Fluorescence microscopy of dye-stained Xenopus XTC-2 nuclei shows that UV illumination has effects similar to chemical fixation by formaldehyde. At 365 nm a dose of approximately 70 J/cm2 results in 50% of the DNA being cross-linked, as measured by chloroform-sodium dodecyl sulfate extraction. At 410 nm the efficiency of cross-linking was smaller by a factor of 3. Gel electrophoresis of the cross-linked proteins shows them to be predominantly core histones. The implications of these results for experiments on live cells stained with H33342, for example, fluorescence microscopy of nuclear dynamics or cell sorting, are discussed.