DNase IIbeta distribution and activity in the mouse lens.
OBJECTIVE
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.
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA. demaria@vision.wustl.edu
Alicia
A
De Maria
Steven
S
Bassnett
D000818:Animals; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D007908:Lens, Crystalline / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D013347:Subcellular Fractions
D012333:RNA, Messenger; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II