Neutral lipids restrict the mobility of broken DNA molecules during comet assays.

Abstract

One widespread technique to assess in relative terms the amount of broken DNA present in the genome of individual cells consists of immobilizing the cell's nucleus under an agarose pad (called the nucleoid) and subjecting the whole genome to electrophoresis to force broken DNA molecules out of it. Since the migrating broken DNA molecules create a tail behind the nucleoid, this technique is named the comet assay. While performing comet assays regularly, we systematically observed circular regions devoid of DNA within the nucleoid region. We characterize here that these correspond to clusters of neutral (apolar) lipids, since they could be labeled with neutral lipid-dying molecules, increased when cells were fed with oleic acid, and were irresponsive to the electrophoretic field. Of relevance, de-lipidation assays, either in vivo, or in vitro using acetone, show that these neutral lipids (NL) within the nucleoid limit the ability of broken DNA molecules to migrate into the comet tail. From a technical point of view, we show that de-lipidation permits a wider range for the detection of broken DNA molecules. Biologically, we put forward the notion that NL in contact with DNA may locally exert regulatory functions within the cell's nucleus.