Detection and Partial Purification of a Cruciform-Resolving Activity (X-solvase) from Nuclear Extracts of Mouse B-Cells

Abstract

We have identified a cruciform-resolving enzyme (X-solvase) in nuclear extracts from mouse B-cells, called EMX1, by using an exonuclease-resistant cruciform DNA as a substrate. The cruciform was a 104-nt oligonucleotide that spontaneously adopted a branched conformation with four arms, each arm protected by a terminal loop of five T residues. A ligatable 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 containing EMX1 show high specificity for cruciform DNA. The cleavage pattern is indistinguishable from that found in the same substrates after treatment with endonuclease VII from phage T4 or endonuclease X3 from the yeast Saccharomyces cerevisiae. EMX1 and yeast endonuclease X3 were also found to be sensitive to anti-(endonuclease VII) antibodies which inhibited their reactions with cruciform DNAs in vitro.