Single-Strand Endonucleases (Origin of Replication/SI Nuclease/Mung Bean Nuclease/M Band) M
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Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2756-2760, July 1977 Biochemistry Release of Escherichia coli DNA from membrane complexes by single-strand endonucleases (origin of replication/SI nuclease/mung bean nuclease/M band) M. ABE*, C. BROWN*, W. G. HENDRICKSON*, D. H. BOYD*, P. CLIFFORDt, R. H. COTE*, AND M. SCHAECHTER* f * Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111; and t Mathematical Institute, Oxford University, Oxford, England Communicated by Gerhard Schmidt, April 22, 1977 ABSTRACT Treatment of gently prepared lysates of Esch- Incubation of Lysate with Endonucleases. Spheroplasts erichia coli with single-strand-specific endonuclease (SI or from (0.025 ml) were added to tubes containing 0.5 ml of buffer (0.1 mung beans) results in the release of about 90% of the DNA from membranes, as determined by the M band technique. The M sodium acetate, pH 5.0/1 mM ZnSO4/0.01 M Mg acetate) released DNA has an average molecular weight of about 1.2 X with heat-denatured calf thymus DNA at 5 Ag/ml. After the 108. addition of 0.01 ml of 5% Sarkosyl (Geigy Chemical Additives Data obtained with endonuclease SI fit a mathematical Co.) and 0.2-10 units of S1 or mung bean endonuclease, the model in which substrate sites are at or near membrane at- tubes were gently rolled to mix and incubated at 450 for 10-20 tachment sites. Data obtained with pancreatic deoxyribonu- min. Shark liver nuclease treatment was for 30 min at 370, and clease or x-rays fit a model for double-strand breaks at random with sites along the DNA. Fitting data to these models, we estimate samples pancreatic deoxyribonuclease (0.01-0.1 ,ug/ml) that there are 18 ± 5 membrane attachment sites. were incubated at 00 for 5 min. The DNA remaining after SI nuclease treatment is enriched Isolation of Membrane-Associated DNA (M Band). for the region near the origin of chromosome replication. Membrane-associated DNA is defined as DNA that sediments Therefore, attachment at this region appears to be chemically with Mg Sarkosyl crystals (M bands). M bands were prepared different from that at the other sites along the DNA. as follows. Samples were transferred by carefully pouring onto 15-47% sucrose gradients containing 0.01 M MgSO4/0.01 M The chromosome of Escherichla coli appears to be attached to Tris, pH 7.6 at 40/0.1 M KCl. Preformed Mg Sarkosyl crystals the cytoplasmic membrane at 20-30 sites (1, 2). The chemical (0.1-0.2 ml), obtained by mixing two parts 0.1 M Mg acetate nature of DNA attachment' to membranes is not known; In an and one part 5% Sarkosyl, were added (without mixing) to the attempt to study the chemical properties of this interaction we sample layer of each gradient. After 15 min, gradients were took as a point of departure the finding that exogenous dena- centrifuged in a Beckman SW 41 rotor at 15,000 rpm for 20-25 tured DNA sticks to membranes under conditions where native min at 4°. Tube contents were separated into a top fraction DNA does not (2, 3). This suggested to us a study of the effect containing all material above the crystal band, and an M-band of treating cell lysates with the single-strand-specific nucleases. fraction containing the crystals themselves. These fractions were We found that such nucleases from Aspergillus oryzae or mung treated with an equal volume of 10% trichloroacetic acid for bean remove DNA from the membrane in a mode that is con- 30 min on ice, filtered through GF/A filters, and washed with sistent with action of the enzymes at or near the membrane- 5% trichloroacetic acid followed by 1% acetic-acid or 95% attachment sites. These results are different from those obtained ethanol before drying and measuring radioactivity in Liqui- by random scission along the DNA. DNA near the origin of fluor/toluene scintillation fluid. replication is not released from membranes by treatment with Sizing of DNA. Samples (0.3 ml) of the treated lysate were SI nuclease. adjusted to 0.2 M NaCl and 1% sodium dodecyl sulfate. [14C]Thymidine-labeled T4 phage were added and the mixture MATERIALS AND METHODS was incubated at 630 for 20 min. The samples were poured Preparation of Spheroplasts. Ten-milliliter cultures of E. gently onto 5-20% linear sucrose gradients containing 0.05 M coli D-10 met (obtained from R. Jayaraman) were grown at sodium phosphate at pH 6.8, 0.1 M NaCl, and 0.05 M 370 in M9 salt glucose medium containing 0.2% casamino acids. Na2EDTA, and spun at 8000 rpm for 16 hr at 200 in an SW 50.1 At 0D600 = 0.15, the culture was labeled with 50-100 ,ul of rotor. Gradients were dripped onto filters (Whatman 3MM, [methyl-3H]thymidine (New England Nuclear Corp.; 1 mCi/ presoaked with 10% trichloroacetic acid), dried, and assayed ml, 6.7 Ci/mmol), and harvested at OD600 = 0.35. Cells were for radioactivity in Liquifluor/toluene scintillation fluid. resuspended in 0.25 ml of 0.1 M Tris-HCl buffer, pH 8.1 at 40, Molecular weights (Mr) were calculated by the method of containing 0.1 M NaCl and 15% sucrose (wt/vol). To this was Freifelder (4) using SI/S2 = (Mri/Mr2?a. The T4 standard added 0.01 ml of 0.25 M Na2EDTA at pH 8.1 and 0.025 ml of was assumed to have a sedimentation coefficient of 60 S and lysozyme (three times recrystallized, Sigma) at 4 mg/ml in 0.01 a molecular weight of 1.2 X 108 (4). The molecular weight of M Tris buffer. After gentle mixing for 3 min at 40, the reaction each fraction of the gradient was determined by its relative was stopped by adding 0.01 ml of 1.0 M Mg acetate, pH 5. distance from the marker. The average number of breaks was Spheroplasts were kept on ice until use. Experiments were done calculated from the equation: with E. coli PC2 dnaC thy- (obtained from Y. Hirota) using n Mr chromosome = f X (proportion cpm in fraction). similar methods. M.. 1 Mr fraction $ To whom reprint requests should be addressed. Enzymes. 51 was obtained from Miles Laboratories or pu- 2756 Downloaded by guest on September 25, 2021 Biochemistry: Abe et al. Proc. Natl. Acad. Sci. USA 74 (1977) 2757 X 20- a~~~~~~~~ 15- 10- i Bottom 10 20 30 40 Top Fraction number 5 10 15 20 25 30 Time, min FIG. 1. Size of DNA after nuclease treatment. Lysates were in- cubated and sized as described in Materials and Methods. A portion FIG. 2. Time course of DNA release from membranes by S1 nu- of each sample was also fractionated on an M band gradient. Results clease. Incubation with nuclease and M band fractionation are de- are similar with different DNA concentrations and when T4 phage scribed in Materials and Methods. Numbers next to curves indicate DNA marker is eliminated. The extrapolated molecular weight for units of enzyme used. the untreated sample is 21 X 109. Similar curves were obtained using mung bean nuclease. 0, Sample incubated without nuclease, 5% of the DNA is in the top fraction ofthe M band gradient; 0, 2 units S1, the membrane. The size of the DNA fragments released from 63% of the DNA is in the top fraction; X, 5 units S1, 85% of the DNA the membrane is relatively constant throughout the time course, is in the top fraction. Arrow indicates position of T4 phage DNA whereas the DNA that remains in the M band decreases to this marker. size limit with increasing enzyme activity (Fig. 3, which shows two of a series of analogous experiments). rified from a-amylase (see below). Mung bean and shark liver Similar results are obtained with other single-strand-specific endonucleases were kindly provided by R. Wells and E. enzymes such as mung bean and shark liver endonucleases. The Goldberg, respectively. Electrophoretically purified pancreatic ability of these enzymes to hydrolyze RNA is apparently not deoxyribonuclease was obtained from Worthington. responsible for release of DNA from the membrane because Purification of SI Endonuclease. A modification of the pretreatment with pancreatic ribonuclease has no effect on the procedure of Vogt (5) was used to purify SI endonuclease from amount of DNA in the M band either with or without SI a-amylase (crude, type IV-A from Aspergillus oryzae) obtained treatment. In a double-label experiment (not shown) it was from Sigma. Purification steps included heat treatment, two found that when the proportion of DNA released from the (NH4)2S04 precipitations, dialysis, DEAE-cellulose chroma- membrane has reached a plateau, the enzyme is fully active tography, and Sephadex G-100 gel filtration. A2j0 and nuclease against newly added lysate and that when a second plateau is activity (using either native or denatured DNA as substrate) reached, both the original and the newly added DNA approach were monitored across the column fractions. the same size limit. The incubation conditions do not reduce Assay of Single-Strand Endonuclease Activity. One or two the activity of Si on single-stranded calf thymus DNA. Release microliters of each fraction was assayed for solubilization of of DNA from the membrane by SI is inhibited strongly by the single- or double-stranded calf thymus DNA by the procedure addition of single-stranded DNA to the lysate but only slightly of Vogt (5). One unit of nuclease activity is the amount of en- by the addition of double-stranded DNA (Fig.