AFM ANALYSIS OF DNA-PROTAMINE COMPLEXES BOUND TO MICA

A novel method for reconstituting sperm chromatin was used to investig ate how protamine 1 condenses DNA. Complexes formed in vitro using lin earized plasmid DNA were imaged and measured by atomic force microscop y (AFM). The structures formed were found to be highly dependent on th e sample preparation method used for reconstitution. Interstrand, side -by-side fasiculation of DNA and toroidal-like structures only 1-2 DNA diameters thick were observed for complexes formed in solution follow ing direct mixing of the DNA and protamine. Large chromatin aggregates were also observed on the mica. However, if the DNA was first allowed to attach to the mica prior to addition of the protamine, well-define d toroidal complexes were formed without any observed DNA fasiculation or aggregate formation. The diameter of the toroids measured 30.6-50. 2 nm (mean 39.4 nm). The dimensions of these structures indicate that the condensed DNA is stacked vertically by four to five turns, with ea ch coil containing as little as 360-370 bp of 'B'-form DNA. This appro ach for preparing and imaging DNA-protamine complexes permits the anal ysis of intermediate structures 'trapped' on the mica as partially for med toruses of nucleoprotamine.