DETECTION AND PARTIAL-PURIFICATION OF A CRUCIFORM-RESOLVING ACTIVITY (X-SOLVASE) FROM NUCLEAR EXTRACTS OF MOUSE B-CELLS

We have identified a cruciform-resolving enzyme (X-solvase) in nuclear extracts from mouse B-cells, called EMX1, by using an exonuclease-res istant cruciform DNA as a substrate. The cruciform was a 104-nt oligon ucleotide that spontaneously adopted a branched conformation with four arms, each arm protected by a terminal loop of five T residues. A lig atable nick was left in one arm. After ligation, the covalently closed substrate was used to follow an 1800-fold purification of the mouse X -solvase (EMX1) from crude nuclear extracts by chromatography on DEAE- cellulose, MonoQ and heparin-Sepharose. The purest fractions containin g EMX1 show high specificity for cruciform DNA. The cleavage pattern i s indistinguishable from that found in the same substrates after treat ment with endonuclease VII from phage T4 or endonuclease X3 from the y east Saccharomyces cerevisiae. EMX1 and yeast endonuclease X3 were als o found to be sensitive to anti-(endonuclease VII) antibodies which in hibited their reactions with cruciform DNAs in vitro.