Abstract
Most known nucleases require a divalent cation as a cofactor, usually Mg2+ or Ca2+, and are inhibited by the chelators EDTA and EGTA. We report the existence of a novel nuclease activity, initially identified in the luminal fluids of the mouse male reproductive tract but subsequently found in the liver, but not in brain, that requires EGTA chelated to calcium to digest DNA. We refer to this unique enzyme as CEAN (Chelated EGTA Activated Nuclease). Using a fraction of vas deferens luminal fluid, plasmid DNA was degraded in the presence of excess Ca2+ (Ca2+:EGTA = 16) or excess EGTA (Ca2+:EGTA = 0.25), but required the presence of both. Higher levels of EGTA (Ca2+:EGTA = 0.10) prevented activity, suggesting that unchelated EGTA may work as a competitive inhibitor. The EGTA-Ca activation of CEAN is reversible as removing EGTA-Ca2+ stops ongoing DNA degradation, but adding EGTA-Ca2+ again reactivates the enzyme. This suggests the possibility that CEAN binds directly to EGTA-Ca2+. CEAN has a greater specificity for the chelators than for the divalent cation. Two other chelators, BAPTA and sodium citrate, do not activate CEAN, but chelated EDTA does. EGTA chelated to other divalent cations such as Mn2+, Zn2+, and Cu2+ activate CEAN, but not Mg2+. The nuclease activity is heat labile as activity is lost upon boiling. Size filtration centrifugation limited the activity to between 30 kDa and 50 kDa in both sperm luminal fluids and liver extracts. These data suggest that EGTA and EDTA may not always prevent DNA from nuclease damage. (poster)
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call