Evidence for dna endonuclease activity in nuclear extracts from mosquito cells

Abstract

We describe a deoxyribonuclease activity from nuclear protein extracts of cultured Aedes albopictus mosquito cells. The nuclease cleaved linear and circular double-stranded DNA, first generating 3′ OH single-stranded nicks followed by second strand cleavage, but had little or no exonucleolytic activity. Detection of this activity was optimal at pH 7.1, in the presence of a divalent cation (Mg 2+, Ca 2+, Mn 2+, Ba 2+). In the presence of Mg 2+, Zn 2+, Hg 2+ and Cu 2+ inhibited activity, sulfhydryl reagents and ATP had no effect. At physiological temperatures (18–35°C), linear double-stranded DNA probes were preferentially cleaved near sites containing 3-6 consecutive deoxyadenine/thymine base pairs. Results from salt dependency and drug inhibition studies, combined with inspection of DNA sequence, suggested that DNA structure is among the parameters that determine preferred cleavage sites.