Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA. demaria@vision.wustl.edu
PURPOSE: To map the cellular and subcellular distribution of DNase IIbeta activity in the mouse lens. METHODS: DNase IIbeta-specific activity was determined by assaying lens lysates prepared from wild-type or DNase IIbeta-null mice. Regional nuclease activity was determined by microdissection of lens samples or a tissue-imprinting assay. Subcellular distribution was determined by density-gradient ultracentrifugation. RESULTS: DNase IIbeta transcripts increased 200-fold in abundance during fiber cell formation, and DNase IIbeta activity accounted for approximately 50% of the acid nuclease activity in the cortical fiber cells. Examination of lenses from DNase IIbeta-null mice confirmed that the enzyme was required for denucleation. In wild-type lenses, nuclei were TUNEL positive before denucleation, indicating that 3'-OH DNA ends had accumulated. However, DNase IIbeta-mediated scission generates 3'-PO(4)(-) DNA ends only. This paradoxical finding was explained by the presence of phosphatases that converted the 3'-PO(4)(-) ends produced by DNase IIbeta into 3'-OH ends. DNase IIbeta activity was strongest early in differentiation, where it was associated with the lysosomal fraction. Later, an increasing proportion of DNase IIbeta activity was found in the cytosol. CONCLUSIONS: DNase IIbeta activity correlated with and was necessary for fiber denucleation and was most likely contained initially within fiber cell lysosomes before release into the cytoplasm.