A calcium-dependent nuclease activity in X. laevis egg extract. (A) CaCl₂, MgCl₂, or MnCl₂ were added to extract at increasing concentrations for 60 min. RNA was isolated from extract, 5′ end-labeled, and run on a denaturing acrylamide gel. (B) Total RNA was isolated from reactions supplemented with no metal, CaCl₂, EGTA alone, or EGTA and CaCl₂ in the same reaction. RNAs were run on denaturing acrylamide gels and stained with SYBR Green II. (C) Precleared egg extract was run over a Heparin HiTrap affinity column, and flow-through was collected. CaCl₂ was added to the flow-through and run over a Heparin HiTrap column preequilibrated with CaCl₂. Proteins were eluted with a salt gradient from 0.1 M to 1 M KCl. Peak protein–containing fractions were pooled and run on a 10% SDS-PAGE gel and Coomassie stained. A band running just under 37 kD in fractions 7 + 8 and 9 + 10 (arrow) was submitted for mass spec analysis and identified XendoU as the putative nuclease. (D) Peak protein fractions from C were run on 10% SDS-PAGE gels containing ³²P-labeled RNA in the resolving portion of the gel. Gels were soaked in buffer to remove SDS and renature proteins, then soaked in buffer containing 1 mM of CaCl₂, MgCl₂, MnCl₂, or no metal overnight at 4°C and imaged by PhosphorImager.