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

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Abstract
Inverted repeat (palindrome) sequences, which are widespread in eukaryotic genomes, probably exist in 2 alternate configurations, a linear form and a cruciform. To investigate the relative frequency of these forms, the DNA of intact mouse tissue culture cells [SVT2] 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 EM 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 was concluded that almost all of the inverted repeat sequences were crosslinked. No significant number of cruciforms was detected by EM. 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. The inverted repeat sequences of the mouse tissue culture cells tested are predominantly in linear forms rather than in cruciform structures inside the cell.

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