Increased nuclease activity in response to high NaCl

Abstract

High NaCl increases DNA breaks. The increase of breaks persists as long as NaCl remains elevated, but rapidly decreases when NaCl is lowered. The mechanism is uncertain. Nucleases are enzymes that break DNA during normal DNA processing (such as replication and transcription), during DNA repair and during apoptosis. In the present studies we tested for activation of nucleases in response to high NaCl. Activities of nucleases in extracts from HeLa cells exposed to 300 and 400 mosmol/kg (NaCl increased) were estimated by single cell electrophoresis (Comet assay). In this assay, broken DNA from cells that are embedded in agarose migrates under the action of an electrophoretic field, forming the "tail" of a "comet". The amount of DNA that migrates out of a nucleus and the distance that it migrates are measures of DNA fragmentation. Cells grown at 300 mosmol/kg were embedded in agarose and permeabilized, then incubated with protein extracts from cells remaining at 300 mosmol/kg or exposed to 400 mosmol/kg for 2 hours. Extracts from cells exposed to 400 mosmol/kg caused a 70% increase in Comet tail moment and doubling of the total Area of the Comet image, indicating increased DNA fragmentation. We propose that activation of nucleases may contribute to the high NaCl‐induced increase of DNA breaks. Supported by the Intramural Program of NHLBI.