Electron microscopy of DNA crosslinked with trimethylpsoralen: test of the secondary structure of eukaryotic inverted repeat sequences

Abstract

It has been suggested that inverted repeat (palindrome) sequences, which are widespread in eukaryotic genomes, exist in two alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells was covalently crosslinked with 4,5',8-trimethylpsoralen (me3-psoralen) in order to prevent rearrangement of the DNA secondary structure during DNA isolation. The distribution of me3-psoralen crosslinks was determined by electron microscopy after denaturation of the DNA in the presence of glyoxal. Because of the high frequency and the relatively uniform distribution of the me3-psoralen crosslinks, it could be concluded that almost all of the inverted repeat sequences had been crosslinked. In spite of this, no significant number of cruciforms was detected by electron microscopy. To determine whether the me3-psoralen might itself be disrupting cruciform structures, cruciforms were first produced in isolated Tetrahymena rDNA by heat treatment and then crosslinked in vitro. The crosslinking was found to stabilize rather than disrupt these cruciforms. We conclude that the inverted repeat sequences of the mouse tissue culture cells we tested are predominantly in linear forms rather than in cruciform structures inside the cell.