Production of Specific Fragments of 4X174replicative Form DNA by a Restriction Enzyme from Haemophilus Parainfluenzae, Endonuclease HP
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JOURNAL OF VROLOGY, Apr. 1973, p. 59689 Vol. 11, No. 4 Copyright @ 1973 American Society for Microbiology Printed in U.S.A. Production of Specific Fragments of 4X174Replicative Form DNA by a Restriction Enzyme from Haemophilus parainfluenzae, Endonuclease HP PAUL H. JOHNSON, AMY SHIU LEE, AND ROBERT L. SINSHEIMER Division of Biology, California Institute of Technology, Pasadena, California 91109 Received for publication 6 November 1972 A restriction endonuclease from Haemophilus parainfluenzae degrades *X174 replicative form DNA into eight specific fragments, ranging from 1,700 to 150 base pairs and terminated specifically by deoxycytidylic acid. Several bacterial restriction deoxyriboendonu- activity eluted after 0.1 M NaCl. The DEAE cleases have been described recently which pro- fractions were stored at 4 C until used. Endo- duce unique fragments from high-molecular- nuclease R was purified as described by Smith weight foreign DNA, are not associated with a and Wilcox (8) and did not include DEAE- known modification activity, and require no cellulose chromatography. special cofactors other than divalent metal ions 'H-OX174am3 replicative form (RFI) DNA (3, 6, 8). These enzymes will be very useful for was isolated from chloramphenicol-treated cells studying DNA structure and function. We are and purified by Biogel chromatography, equi- interested specifically in the use of sequence- librium centrifugation in CsCl-ethidium bromide specific deoxyriboendonucleases for the isolation density gradients, and sedimentation in isokinetic and nucleotide sequence determination of the neutral sucrose gradients. Details of the isolation initiation sites for DNA replication and transcrip- procedure will be described elsewhere. tion, and for the analysis of 4X174 replicating Electrophoresis of DNA fragments was per- intermediates. formed in 5% polyacrylamide gels (1 by 20 cm) Endonuclease HP was isolated from cultures of formed in Plexiglas tubes. The buffer and Haemophilus parainfluenzae (strain originally methods used were those described by Loening from Sol Goodgal and G. Leidy) by using a (5) plus 0.1% (wt/vol) SDS. Electrophoresis was modification of -the procedure developed by performed at room temperature at a constant Smith and Wilcox (8) for the purification of potential of 45 V; running time was 1 h per cm of endonuclease R from H. influenzae. The enzyme gel length. To quantitate the radioactivity, gels eluted from a Biogel column (2.5 by 48 cm) be- were frozen and cut into 1-mm segments by using tween volumes of 180 and 230 ml. Fractions the Mickle gel slicer (Brinkman Instruments). containing endonuclease activity and A230/ Gel slices were solubilized with toluene-Liquifluor Ano > 3 were pooled. After the ammonium containing 10% NCS (Amersham-Searle), and the sulfate precipitation (8), the enzyme preparation radioactivity was measured in a Beckman liquid was desalted by chromatography on Sephadex scintillation counter. G-25. The enzyme was then eluted from a End-group determination was performed as phosphocellulose column at 0.2 M KCl. described in the legend to Table 2. Polynucleo- Contaminating exonuclease activity present tide kinase was purified from T4 phage-infected at this stage of the purification was removed cells by using Richardson's original procedure (7) by chromatography on DEAE-cellulose (What- modified by employing DNA-cellulose and Biogel man DE52). For this purpose, a column was exclusion chromatography. Details will be pub- equilibrated with 0.01 M potassium phosphate lished elewhere. buffer, pH 7.4, and the enzyme was applied of an endo- at 10 mg of protein per ml of column bed volume. Figure 1 shows the fractionation Elution was stepwise with three column volumes nuclease HP limit digest of jX174 RF DNA by each of 0.05 M, 0.1 M, and 0.2 M NaCl in 0.01 M 5% polyacrylamide gel electrophoresis. Improved potassium phosphate, pH 7.4. Endonuclease HP separation of fragments P1 and P2 can be ob- eluted at 0.05 M NaCl, whereas the exonuclease tained by using gels of lower acrylamide concen- 596 VOL. 11, 1973 NOTES 597 40 30 20 _ b-x 10 0 4 Q-0 E 0- E 5 4 *\ " \\0= 3 2 m=I 20 40 60 80 100 Mobility-._ Mobility -_ FIG. 1. Polyacrylamide gel electrophoresis of FIG. 2. Relationship between mass and electro- *OX174 RF DNA fragments produced by endo- phoretic mobility for OX174 RF DNA fragments nuclease HP. The reaction mixture consisted of produced by cleavage with endonuclease HP. The 0.1 ug of 'H-RFI, 7mM Tris, 7 mM mercaptoethanol, mass is assumed to be proportional to the total num- 50 mM MgCl2, pH 7.4; total volume, 100 pliters. A ber of counts in each peak and is plotted against O-pliter amount of endonuclease HP was added at the distance migrated during the experiment (mo- zero time and again at h. Digestion was at 37 C bility). The parallel line (---) is that calculated for 10 h. To stop the reaction, EDTA ws added to for a peak with twice the mass; the point on this line 0.01 M. Sucrose and sodium dodecyl sulfate were represents P4. added to 5 and0.5% final concentrations, respectively. The mixture was incubated at 37 C for 10 min and DNA layered on a 6% polyacrylamide gel column (1 T.iBLE 1. Size of OX174 RF fragments pro- by 20 cm). Electrophoresis was as described in the duced by cleavage with endonuclease HP text. Mobility is the distance of migration in milli- Fragment no.a Nucleotide pairsb meters at the end of the run. P1 .......................... 1,670 tration. The positions and proportioins of the P2.......................... 1,400 peaks do not change with increasing incubation P3.......................... 825 time or increasing enzyme concentration, indi- P4.1, 4.2.......................... 470 cating that a limit digest of the DNA has been P5.......................... 330 produced. P6...................... 180 Figure 2 shows that there is a linear relation- P7 ...: ............................ 150 ship between the logarithm integrated of the a Fragment numbers correspond to those indi- counts in each peak (Fig. 1) and the electro- cated in Fig. 1. phoretic mobility. Fragment P4 cointains twice b The niumber of nucleotide pairs is calculated the mass expected, suggesting the presence of two by assuming that the total is equal to 5,500 and distinct fragments of similar size. A partial that mass is proportional to the total number of separation of these two fragments can be ob- counts in each peak. tained with longer electrophoresis time. Table 1 shows the size of the endonuclease mined by electronl microscopy and sedimenta- HP fragments calculated from the fraction of tion velocity analysis (1, 2). The size and number total counts present in each peak. It has been of fragments produced by endonuclease HP are demonstrated that the molecular weights of distinct from those produced by the restriction double-stranded DNA fragments determined endonuclease from H. influenzae, endonuclease by mobility during polyacrylamide gel electro- R. We have found that the latter enzyme pro- phoresis are in good agreement with values deter- duces at least 12 specific fragments from 6X174 598 NOTES J. VIROL. TABLE 2. A comparison of the 5'-terminal nucleo- RF DNA which ranlge ini size from 1,200 to 100 tides of OX174 RF DNA fragments produced by base pairs, in close agreement with the pub- endonuclease HP and endonuclease Ra lished data of Edgell et al. (2). Chemical evidence for a difference in the recog- Mol % nucleotide nition site for endonuclease HP and endonuclease Substrate Restrictionenzyme at 5' ends R is presented in Table 2. The nucleotide se- quence of the recognitioii site for endonuclease A T G C R was determined to be G T Py I Pu A C (4) *X174 RFI Endo HP <1 <1 <1 97 where the center dinucleotide is ambiguous and #X174 RFI Endo R 55 6 35 4 the arrow indicates the point of cleavage. T7b Endo It 60 0 40 0 We find coinsistent results with qX174 RF DNA Lambda 44 7 45 4 as substrate for endonuclease R (Table 2). The enzyme preparation had a very low, but detect- a OX RF DNA (>98% form I) was degraded by able, level of exonuclease contamination which endonuclease HP in a reaction mixture consist- might account for the production of 10% pyrimi- ing of 7 mM Tris (pH 7.5), 7 mM mercaptoethanol, dines. The data indicate that the recognition site 50 mM MgCl2, 50 mM NaCl, 12 ug of DNA, 125 for endonuclease HP is different since only deoxy- uliters enzyme (0.1-0.5 units); total volume, 150 Aiters. Incubation was at 37 C for 14 h. The reac- cytidylic acid is found at the 5' ends of frag- tion with endonuclease R was the same except ments produced by this enzyme. The presence of the MgCl2 concentration was 7 mM. The reaction a single, unique nucleotide at the 5' ends of these was stopped by incubation at 0 C with twofold fragments suggests that the recognition site for excess EDTA. The reaction mixture was adjusted endonuclease HP may not contain an ambiguous to 0.1 M Tris (pH 8.0) and 1 unit of alkaline phos- dinucleotide as does the site for endonuclease R; phatase (Worthington; purified free from endo- however, we do not regard this demonstration nuclease activity) per ml in a volume of 200 as rigorous until the terminal nucleotides have Aiters, and incubated at 37 C for 45 min. The been analyzed for each fragment. phosphatase was inactivated by addition of ethylene-glycoltetraacetic acid to 6.5 mM fol- at This investigation was supported by Public Health lowed by incubation for 7 min 65 C (Theodore Service grant GM 13554 from the National Institute of Live, personal communication). The reaction General Medical Sciences.