Direct Inhibition of Nuclease Activity by KCl
Because it was possible that the potassium-mediated inhibition of internucleosomal DNA cleavage in isolated granulosa cell nuclei could be indirect via a suppression of nuclear protease activity, two final experiments were conducted to further examine the possible direct actions of KCl on nuclease activity. Consistent with data recently reported using a cell-free assay to monitor granulosa cell nuclease activity, nuclear protein extracts prepared from nona-poptotic granulosa cells catalyzed cleavage of linearized plasmid DNA (Fig. 8). However, inclusion of 150 mM KCl, NaCl, or LiCl in the reaction mixture completely suppressed nuclease activity present in the granulosa cell nuclear protein extracts (Fig. 8). Because all nuclease activity in this assay was suppressed by potassium, it may be that the large-fragment cleavage enzyme was not efficiently extracted from the granulosa cell nuclei or that the large-frag-ment cleaving nuclease requires DNA to be in a chromatin conformation in order to be active.
These data were reinforced by the results of the second set of experiments, in which the actions of KCl, NaCl, and LiCl were assessed in a simple system containing only ions, purified nucleases, and naked linearized plasmid. Incubation of plasmid DNA with either DNase-I (Fig. 9, A and B) or DNase-II (Fig. 9, C and D) resulted in a rapid degradation of the plasmid, with the extent of degradation dependent upon amount of enzyme used (Fig. 9, B and D) and the length of reaction (data not shown). At low levels of added nuclease (0.01 U/reaction), inclusion of 150 mM KCl, NaCl, or LiCl in the reaction completely suppressed plasmid DNA degradation (Fig. 9A; data not shown for 0.01 U DNase-II), and these effects were mimicked by the general nuclease inhibitor SAM (Fig. 9A).
FIG. 8. Potassium-mediated suppression of plasmid DNA degradation catalyzed by granulosa cell nuclear protein extracts. Representative photograph depicting a conventional agarose gel electrophoretic analysis of linearized plasmid DNA (intact plasmid is indicated by the arrow) after incubation with vehicle (control, CON) or with granulosa cell nuclear protein extracts in the absence or presence of 150 mM KCl, NaCl, or LiCl. Note the smear of DNA degradation products generated by the nuclear protein extract nuclease or nucleases and the inhibition of plasmid degradation by the presence of KCl, NaCl, and LiCl.
FIG. 9. Effects of potassium on plasmid DNA degradation catalyzed by purified DNase-I or DNase-II. A) Linearized plasmid DNA was incubated without nuclease (-DNase I) or with 0.01 U of DNase-I in the absence (CON, control) or presence of 150 mM KCl, NaCl, or LiCl or with 1 mM SAM. The reactions were then subjected to CAGE, and gels were stained with ethidium bromide to visualize the DNA. Note that the linearized plasmid (indicated by the arrow) is cleaved by DNase-I and that this is prevented by inclusion of KCl, NaCl, LiCl, or SAM in the reaction. Similar data were obtained with 0.01 U of DNase-II (not shown; see D for KCl data). B) Reactions were prepared as described in A, with the exception that increasing amounts of DNase-I were mixed with plasmid DNA in the absence (CON) or presence of a fixed concentration of KCl (150 mM). The ability of KCl to suppress nuclease activity was lost with increasing amounts of nuclease added. C) Plasmid DNA degradation reactions were prepared as described in A using 0.1 U of DNase-II in place of DNase-I. Note that despite the fact that SAM maintains the ability to inhibit plasmid DNA degradation catalyzed by this level of DNase-II activity, KCl, NaCl, and LiCl are without effect. Similar data were obtained with 0.1 U of DNase-I (not shown; see B for KCl data). D) Linearized plasmid was incubated with increasing amounts of DNase-II, as described for DNase-I in B, in the absence or presence of 150 mM KCl. Similar to the case of results obtained with DNase-I (B), the ability of KCl to suppress DNase-II activity was lost with higher amounts of the nuclease.