SEQUENCE-SPECIFIC INTERACTION OF DNA AND CHROMOSOMAL PROTEIN

The principles which regulate nucleotide sequence-specific binding of chromosomal protein to DNA have been studied with isolated chromatin. The experimental strategy is to dissociate chromosomal proteins from chromatin by 2 m-NaCl and then to determine the conditions under which the chromosomal proteins can be caused to reassociate with DNA at sites the same as, or equivalent to, those to which they were originally bound. Similarity or difference of reconstituted and native chromatin is determined by hybridization competition for denatured DNA between RNA transcribed from native chromatin and that transcribed from reconstituted chromatin. It is shown that when chromosomal proteins, principally histones, are dissociated from DNA by 2 m-NaCl and then reassociated by gradient dialysis to low ionic strength, the original chromatin is not reconstituted. Reconstitution by gradient dialysis from 2 m-NaCl in the presence of 5 m-urea followed by removal of the urea does result in chromatin equivalent to native by the hybridization-competition criterion. At the temperature and salt concentrations used, 5 m-urea constitutes a condition under which RNA hybridizes to DNA. For such sequence-specific reconstitution of chromatin the participation of chromosomal RNA is required. © 1969.