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A.B. DeMaria, S. Bassnett; DLAD Activity in Lens Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1990.
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DLAD plays a key role in chromatin degradation in differentiating lens fiber cells (Nishimoto et al., Nature, 2003). To better understand this process, we examined DNA fragmentation and acid nuclease activity in wild type and DLAD knockout mouse lenses.
TUNEL labeling, with and without alkaline phosphatase (CIAP) pretreatment, was used to determine the presence of DNA free 3’PO4 and 3’OH ends, respectively, during fiber differentiation. Nuclease activity in cell lysates was examined using lambda DNA as substrate, and quantified with FluorChemSP software. To visualize DLAD activity in situ, we developed a novel tissue imprinting assay in which nuclease diffusing from cryosections digests an immobilized DNA substrate.
In contrast to wildtype lenses, degenerating fiber cell nuclei were not TUNEL positive in DLAD null lenses, suggesting that DLAD might be the only nuclease responsible for chromatin degradation. Treatment of wild type lenses with CIAP did not resolve in increased TUNEL labeling. We hypothesized that this may reflect the activity of an endogenous phosphatase that rapidly eliminates PO4 groups. Consistent with this notion, the addition of a phosphatase inhibitor to in tube tailing assays resulted in a reduction in the number of labeled 3’OH ends. In vitro and tissue imprinting assays demonstrated that DLAD was present in the fibers. The activity level was highest in cortical cells and lowest in nuclear fibers. Maximal activity was observed at acidic pH (pH 5.9) but some activity was also noted at pH 6.8 (cytoplasmic pH in the innermost fiber cells).
DLAD plays a critical role in DNA degradation during fiber differentiation. It is found in cells with intact chromatin, implying the presence of a regulatory mechanism that prevents premature DNA degradation. Understanding DLAD regulation may help explain the mechanism by which nuclei and other organelles are eliminated during fiber differentiation.
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