A novel nuclease activity that is activated by Ca(2+) chelated to EGTA.
Most nucleases require a divalent cation as a cofactor, usually Mg(2+) or Ca(2+), 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 other tissues,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 Ca(2+) (Ca(2+) :EGTA = 16) or excess EGTA (Ca(2+) :EGTA = 0.25), but required the presence of both. Higher levels of EGTA (Ca(2+) :EGTA = 0.10) prevented activity, suggesting that unchelated EGTA may be a competitive inhibitor. The EGTA-Ca(2+) activation of CEAN is reversible as removing EGTA-Ca(2+) stops ongoing DNA degradation, but adding EGTA-Ca(2+) again reactivates the enzyme. This suggests the possibility that CEAN binds directly to EGTA-Ca(2+). CEAN has a greater specificity for the chelator than for the divalent cation. Two other chelators, BAPTA and sodium citrate, do not activate CEAN in the presence of cation, but chelated EDTA does. EGTA chelated to other divalent cations such as Mn(2+), Zn(2+) , and Cu(2+) activate CEAN, but not Mg(2+) . The activity is lost upon boiling suggesting that it is a protein. These data suggest that EGTA and EDTA may not always protect DNA from nuclease damage.
Dominguez
Kenneth
K
Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96822, USA.
Ward
W Steven
WS
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002614:Chelating Agents / Q000494:pharmacology*; D003851:Deoxyribonucleases / Q000302:isolation & purification* / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D004789:Enzyme Activation; D008297:Male; D051379:Mice; D014018:Tissue Distribution
D002614:Chelating Agents; D004533:Egtazic Acid; D003851:Deoxyribonucleases; C547277:chelated EGTA activated nuclease, mouse; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium