Identification of a potent decatenating enzyme from Escherichia coli.

R J Digate,K J Marians

doi:10.1016/s0021-9258(18)37713-5

Abstract

A topoisomerase has been purified from extracts of a topoisomerase I-deficient strain of Escherichia coli based solely on its ability to segregate pBR322 DNA replication intermediates in vitro. This enzyme rapidly decatenated multiply linked form II:form II DNA dimers to form II DNA, provided that the DNA substrate contained single-stranded regions. Efficient relaxation of negatively supercoiled DNA was observed when reaction mixtures were incubated at 52 degrees C, but not at 30 degrees C (the temperature at which decatenation was readily observed). This topoisomerase was insensitive to the DNA gyrase inhibitor norfloxacin and unaffected by antibody directed against topoisomerase I. Relaxation of a unique plasmid topoisomer revealed that this decatenase changed the linking number of the DNA in steps of one and was therefore a type 1 topoisomerase. The cleavage pattern of a fragment of single-stranded phi X174 DNA generated by this decatenase was virtually identical to that reported for topoisomerase III, the least characterized topoisomerase present in E. coli.

Highlights

Three topoisomerases have been purified from Escherichia coli
The addition of Top0 I (Minden and Marians, 1986) or small amounts of a soluble extract prepared from wild-type E. coli t o the replication reaction mixture (Minden and Marians, 1985; Fig. l A, lanes 5-7) resulted in the segregationof t h e DNA replication intermediates intoform I DNA
Solubleextracts prepared from the E. coli strain DM700, which lacks Top0 I activity, containedan activity that could catalyze the segregation of pBR322 DNA replication intermediates(Fig. 1A, lanes 24)

Summary

Identificationof a Potent Decatenating Enzyme froEmscherichia coli*

The DNA pellet was resuspended in 50 mM Tris-HC1 buffer (pH 8.8 at 37 "C), 10 p~ ZnSO,, pg/ml bovine serum albumin and the 5' termini dephosphorylated by incubation with bacterial alkaline phosphatase (0.75unit) at65 "C for 1h This reaction mixture was extracted twice with pheno1:chloroform and theDNA precipitated with ethanol. Relaxation of Superhelical DNA-Reaction mixtures (25 pl) containing 40 mM Hepes-KOH buffer (pH 8.0 at 25 "C),1mM magnesium acetate, 10 mM dithiothreitol, 100 pg/ml bovine serum albumin, 40% v/v glycerol, and 200 ng of pBS-M13 form I DNA were incubated as described in the figure legends. Topoisomerase Cleavageof Single-stranded 4x174DNA-Reaction mixtures (0.1 ml) contained 50 mM Hepes-KOH buffer (pH 8.0 at 23 "C), 5 mM dithiothreitol, 100 pg/ml bovine serum albumin, 40% glycerol,32P-end-labeledAuaII-PsDtINA fragment (55,000cpm), and either 900 units of Top0 I or 125 units of the decatenase. The samples were heated at 90 "C for 1 min and electrophoresed through a 12% polyacrylamide gel (30 X 31 X 0.4 cm) containing 8 M urea as described by Maxam and Gilbert (1980).The gel was fixed, dried, and autoradiographed

RESULTS

The decatenase is probably not related to DNA gyrase or

DISCUSSION