Proc. Natl. Acad. Sci. USA Vol. 73, No. 12, pp. 4324-4328, December 1976 Biochemistry Endonuclease II, apurinic acid endonuclease, and exonuclease III (chemical carcinogens/DNA repair/Escherichia coli endonucleases and exonucleases) D. M. KIRTIKAR, G. R. CATHCART, AND D. A. GOLDTHWAIT Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106 Communicated by Harland G. Wood, August 23, 1976 ABSTRACT An endonuclease of Escherichia coli active on They found that the two activities copurified and a thermo- a DNA treated with methylmethane sulfonate has been sepa- rated from an endonuclease active on depurinated sites. The sensitive mutant of one activity was thermosensitive for the former enzyme is designated here as endonuclease II, while the other activity. Furthermore, in a revertant of one of the mu- latter enzyme is designated as apurinic acid endonuclease. tants, the levels of both activities increased. This evidence Endonuclease II is also active on DNA treated with- methylni- suggested that the two enzyme activities were due to the same trosourea, 7-bromomethyl-12-methylbenz[a anthracene, and protein. Weiss has recently purified a protein of molecular 'y-irradiation. A third fraction which contains activities for both weight 28,000 to homogeneity (13) and showed that it has the depurinated and alkylated sites needs further study. Endonu- clease II, molecular weight 33,000, has been purified 12,500-fold activity expected of exonuclease III and also has an activity on and does not have exonuclease III activity. Apurinic acid en- the heavily alkylated DNA entrapped in polyacrylamide gel. donuclease, molecular weight 31,500, has been purified He has concluded that endonuclease II of E. coil is exonuclease 11,000-fold and does not have exonuclease III activity. Exonu- III. However, we have shown that the three enzymes, endo- clease III, molecular weight 26,000, has been purified 2300-fold nuclease II, exonuclease III, and apurinic acid endonuclease, and does not have endonucleolytic activity at depurinated re- are separate proteins. Furthermore, we have noted that a pu- duced sites or at alkylated sites in DNA. Therefore, these are rified preparation of exonuclease III can make three separate proteins. Exonuclease III can produce, presum- double-strand ably by its exonucleolytic activity, double-strand breaks in breaks in heavily alkylated DNA under conditions where it is heavily alkylated DNA under conditions where it does not make unable to make single-strand breaks at alkylated or depurinated single-strand endonucleolytic breaks at either depurinated- sites. reduced or alkylated sites. METHODS The first purpose of this paper is to define endonuclease II of Assays. The assays for the endonucleases involved either Escherichia coli as an activity different from the apurinic acid DNA immobilized in acrylamide gel or DNA examined on endonuclease of E. coli. Strauss and Robbins first described an sucrose gradients. [3HjThymidine-labeled T4 DNA was pre- endonucleolytic activity in extracts of Bacillus subtilis that pared as described (2). Preparations of DNA had specific ac- recognized alkylated DNA (1). In this laboratory, an enzyme tivities ranging from approximately 1 to 5 X 103 cpm/nmol. in extracts of E. coli, active on heavily alkylated DNA, was Endonuclease assays, using either the DNA-gel or sucrose partially purified, characterized, and designated endonuclease gradients, and the exonuclease assay are described in the leg- II of E. coli (2, 3). The substrate used for these experiments was ends to Figs. 1 and 2. DNA that was entrapped in a polyacrylamide gel and then al- Enzymes. The E. coli strain used for small-scale enzyme kylated with methylmethane sulfonate [MeSO2OMe (MMS)] purification was AB 1157. This was grown in a fermentor to at an MeSO2OMe-to-nucleotide ratio of 6000 to 1. A partially mid-logarithmic phase in a modified EM-9 medium (16) sup- purified preparation of endonuclease II was also found to have plemented with L-leucine, L-proline, L-histidine, L-threonine, an endonucleolytic activity on depurinated reduced DNA (4), and L-arginine, each at 20 Asg/ml. For the initial fractionation, and this activity was thought to be due to the same enzyme that see legend of Fig. 1. was active on MeSO20Me-treated DNA. However, Verly et For a large-scale preparation of the apurinic acid endonu- al. (5, 6), using the purification procedure originally described clease and endonuclease II, E. coli JC4583 (endo I-, his-, F-, in this laboratory, obtained an enzyme that was active on gal-, SMs, BI-) was grown at the New England Enzyme Center, depurinated DNA but not on alkylated DNA. Subsequently, and 800 g were used. The purification steps will be described we succeeded in separating the activity on depurinated sites in future publications. Although each purified endonuclease in DNA from the activity on MeSO2OMe-treated DNA (7, 8). preparation, when examined by sodium dodecyl sulfate (Na- The former we designate as the apurinic acid endonuclease of DoSO4)-gel electrophoresis, showed only a single band, it could E. coli, while the latter we designate as endonuclease II of E. not be concluded unequivocally that the preparations were coli. Endonuclease II of E. coli is also active on DNA treated homogeneous because large amounts of enzyme were not with methylnitrosourea, 7-bromomethyl-12-methvlbenz- available to look for small amounts of contaminant protein. [alanthracene, and y-irradiation (7-11). Exonuclease III was purified from 200 g of E. coil (JC 4583) The second purpose of this paper is to demonstrate that en- by a modification of the procedure of Richardson and Kornberg donuclease II, the apurinic acid endonuclease, and exonuclease (15), which included ammonium sulfate fractionation and III are separate proteins. Originally, Yajko and Weiss (12) DEAE-cellulose, phosphocellulose, Sephadex G-100, and hy- demonstrated that a number of E. coli mutants deficient in droxyapatite column chromatography. The enzyme purifica- exonuclease III were also deficient in "endonuclease II" and tion was 2300-fold when the 3'-phosphatase activity in fraction vice versa. The "endonuclease II" activity was measured with III due to exonuclease III was used for the calculation. The heavily alkylated DNA in acrylamide gel (2), as noted above. preparation was not homogeneous by NaDodSO4 gel electro- phoresis. Dr. C. C. Richardson very kindly provided a sample Abbreviations: MeSO2OMe, methylmethane sulfonate (MMS); Na- of his purified exonuclease III for comparison with this prep- DodSO4, sodium dodecyl sulfate. aration. 4324 Downloaded by guest on September 25, 2021 Biochemistry: Kirtikar et al. Proc. Nati. Acad. Sci. USA 73 (1976) 4325 0 N K b E E 0. 0. U Pt 01 FIG. 1. Chromatographic behavior of apurinic acid endonuclease, endonuclease II, and exonuclease III on DEAE-cellulose. Fraction III (2), a 45-75% ammonium sulfate precipitate (396 mg), was dialyzed against buffer C (0.05 M Tris.HCl at pH 8.0, 0.1 mM dithiothreitol, and 20% glycerol) and then applied to a DE-52 column 2.5 X 48 cm. Elution was with one column volume of buffer C plus 0.03 M NaCl and then as indicated in the figure. Ten-milliliter fractions were collected and 50 AI ofevery fourth fraction was assayed for apurinic acid endonuclease, endonuclease II, and exonuclease III. For the endonuclease assays, labeled DNA was entrapped in a polyacrylamide gel which was forced through a screen to produce gel particles (14). Depurinated reduced DNA gel was prepared by suspending the gel in four volumes of 0.1 M sodium citrate buffer at pH 3.5 plus 0.1 mM EDTA and heating at 450 for 30 min. The gel was then cooled, the pH was adjusted to 6.5 with NaOH, and potassium phosphate buffer at pH 6.5 was added to a final concentration of 0.5 M. Aldehyde groups at depurinated sites were reduced with NaBH4 (4) to prevent spontaneous a-elimination with phosphodiester bond hydrolysis. A final concentration of 0.25 M NaBH4 was attained by three additions at 15-min intervals at room temperature. After an incubation of 60 more min, the DNA gel was washed in 0.05 M Tris-HCl at pH 8.0 and resus- pended in the same buffer. These conditions produce approximately one depurinated site per 1550 nucleotides. The MeSO2OMe-treated DNA gel was prepared by incubation of the DNA gel for 120 min at room temperature in 0.05 M Tris-HCl at pH 8.0 with MeSO2OMe at a molar ratio of MeSO2OMe to DNA nucleotide of 500 to 1. Gels were washed extensively with 0.05 M Tris.HCl at pH 8.0 and resuspended in the same buffer. The packed DNA gels contained 60-80 nmol of DNA nucleotide per ml of packed volume. Both types ofgels were used on the day of their prep- aration. Incubation mixtures contained 20 nmol of DNA substrate, 0.05 M Tris-HCl at pH 8.0, 10-4 M 8-hydroxyquinoline, 10-4 M dithiothreitol, and 1.5 mg of bovine serum albumin in a volume of 1.5 ml. Incubations were at 37° for 30 min and were stopped with 0.1 ml of 1% NaDodSO4. After dilution with water to 2.0 ml and centrifugation, the radioactivity of a 1.0-ml aliquot was determined in a liquid scintillation counter. One unit represents 1 ,umol of DNA nucleotide released per hr. (0) Depurinated reduced [3H]DNA gel; (0) MeSO2OMe-treated [3H]DNA gel. Exo- nuclease III was assayed by its 3'-phosphatase activity. E. coli [32P]DNA (2 X 104 cpm/nmol) was digested with micrococcal nuclease until 30% was acid soluble; the higher-molecular-weight material remaining after dialysis was used (15). Incubation mixtures contained 50 nmol of DNA, 10-3 M 2-mercaptoethanol, 10-2 M MgCl2, and 0.066 M potassium phosphate at pH 7.0 in a volume of 0.3 ml.