Numerous studies in various animal models have demonstrated that fiber denucleation is preceded by DNA strand scission and accumulation of 3′-OH DNA termini.
1 3 4 19 20 The presence of nuclei in the central fiber cells of DNase IIβ-null lenses indicated that DNase IIβ was absolutely required for normal denucleation. Paradoxically, however, DNase IIβ digestion results in the production of 3′-PO
4 − DNA ends rather than 3′-OH.
14 To reconcile these apparently contradictory observations, the integrity of nuclear DNA was analyzed in differentiating Wt or DNase IIβ-null fiber cells by using a modified TUNEL assay
(Fig. 6) . In this assay, 3′-OH ends were identified using fluorescein-labeled dUTPs with or without alkaline phosphatase pretreatment. The TUNEL assay confirmed the presence of 3′-OH DNA ends in Wt fiber cells bordering the OFZ
(Fig. 6B) . In contrast, no 3′-OH labeling was observed in DNase IIβ-null lenses
(Fig. 6D) . This observation indicated that DNase IIβ activity was required for the accumulation of 3′-OH ends in the disintegrating fiber nuclei. Wt lens sections pretreated with alkaline phosphatase before TUNEL labeling were not labeled more strongly than untreated cells (compare
Fig. 6Bwith 6F), suggesting that the number of 3′-PO
4 − ends present was negligible compared with the number of 3′-OH ends. A plausible explanation for these observations was that during fiber denucleation, DNase IIβ activity generates DNA fragments with 3′-PO
4 − ends but that these are subsequently (and rapidly) converted to 3′-OH ends by the action of endogenous phosphatases. To test this hypothesis, we developed an in tube labeling assay that used a nuclease activity assay
(Fig. 6I)in conjunction with a 3′-OH end labeling reaction
(Fig. 6J) . Cortical lysates from Wt lenses were mixed under acidic conditions with an exogenous DNA substrate, resulting in degradation of the substrate
(Fig. 6I) . The 3′-OH ends in the degraded DNA were then labeled using the Klenow fragment. The addition of a broad-spectrum phosphatase inhibitor to the lysate had no effect on the extent of DNA degradation
(Fig. 6I)but reduced the number of labeled 3′-OH ends significantly
(Figs. 6J 6K) . These data supported the notion that DNA degradation was accomplished through the actions of DNase IIβ that cut the DNA to produce 3′-PO
4 − ends, which were converted subsequently to 3′-OH ends by an endogenous phosphatase(s).