Induction of proteinase K-sensitive sites and M. luteus endonuclease-sensitive sites in DNA of barley embryos by sodium azide

Abstract

DNA damage induced in germinating barley embryos by mutagenic and sublethal doses (0.1–2 mM, 2 h) of sodium azide, applied at pH 3, was measured by alkaline elution. Isolated nuclei were lysed at a high pH with either 2% SDS or 2 M NaCl on polyvinyl chloride filters and digested with proteinase K or with Micrococcus luteus endonuclease prior to elution. The azide treatments resulted in a dose-dependent increase of proteinase K-sensitive sites and an appearance of Micrococcus luteus endonuclease-sensitive sites. These sites were detected as DNA single-strand breaks after digestion of the DNA with either one or both of the enzymes. The two types of lesion were additive and occurred in a ratio of about 1:1. The additive effect suggested independent origin for the two types of lesion. Breaks independent of proteinase K digestion appeared only when DNA was analysed 24 h after the action of azide. The nature and significance of these DNA lesions are discussed.