PAUL H. JOHNSON,* MICHAEL J. MILLER, ELIZABETH WILD, SAMUEL V. KELLY, AND. LAWRENCE I. GROSSMANt. Department ofBiochemistry, Wayne State ...
JOURNAL OF VIROLOGY, Nov. 1979, p. 629-639 0022-538X/79/11-0629/11$02.00/0
Vol. 32, No. 2
Electrophoretic Characterization of Intracellular Forms of Bacteriophage 4X174 DNA: Identification of Novel Intermediates of Altered Superhelix Density PAUL H. JOHNSON,* MICHAEL J. MILLER, ELIZABETH WILD, SAMUEL V. KELLY, AND LAWRENCE I. GROSSMANt Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201 Received for publication 15 March 1979
The replication cycle of bacteriophage 4X174 DNA has been analyzed by agarose gel electrophoresis. The electrophoretic behavior of the predominant species of parental and progeny DNA molecules formed between 5 and 40 min after infection was deduced and quantitated. Migration through 1.4% agarose at 5 and 10 V/cm resolved all known viral DNA species as well as fragments of host chromosomal DNA. Among parental replicative form (RF) molecules synthesized, 1 to 3% were full length linear duplexes (RFIII) and approximately 65% were closed circular duplexes (RFI). Most of the input viral strands remained in a duplex structure throughout the period of infection studied here. Among progeny molecules, RFIII was not readily detected unless viral DNA synthesis was inhibited by chloramphenicol. Late in infection, 20% of the progeny RF were found to exist as form I DNA. In addition, approximately 1% of the viral DNA was found as unit length linear single strands. Electrophoretic analysis of RF DNA after controlled denaturation suggests the existence of four populations of closed circular RF: (i) molecules of native superhelix density (RFI); (ii) a population of molecules of altered topological linking number, a, differing in increments of one superhelical turn (T) between T values of 0 and approximately -31; (iii) a superimposed population of topological isomers which under electrophoresis conditions have a mean T value (T) equal to +5; and (iv) a population of "complexed" molecules with a reduced number of superhelical turns due to their association with single-stranded DNA and RNA. Complexed parental molecules isolated from cells infected at high multiplicity release RFI and homologous single-stranded DNA upon denaturation and are postulated to be intermediates in genetic recombination. Complexed RF DNA isolated from cells infected at low multiplicity release native supercoils upon reaction with RNase H and are observed by electron microscopy to contain displacement loops. Such molecules are likely intermediates in transcription. Our results are consistent with a structure of complexed RFI involving a partially triple-stranded helix in which a covalently closed circular duplex molecule contains a reduced number of superhelical turns due to the unwinding produced by base pairing between one strand of the supercoil and an associated homologous single strand of DNA or RNA. The molecular transformations which accompany the infection of animal and bacterial cells by lytic viruses generate a substantial number of discrete intermediate viral DNA species. The difficulty in detecting and separating many of these forms has inhibited the precise delineation of their structures and roles in the viral life cycle. Hydrodynamic methods of analysis are frequently used in the isolation and characterization of viral DNA; however, these techniques
are often not sufficiently powerful to detect and resolve DNA molecules of similar conformation or species present in low amounts. Consequently, we have developed analytical gel electrophoretic techniques for the purpose of studying DNA structure and protein-DNA interaction mechanisms. A previous publication from this laboratory (24) characterized the electrophoretic behavior of the closed circular, nicked circular, linear duplex, and denatured forms of several DNA species in the molecular weight range of 3 x 106 to 11 x 106. We demonstrated that alterations in electric field strength, gel concentra-
t Present address: Department of Cellular and Molecular Biology, Division of Biological Sciences, University of Michigan, Ann Arbor, MI 48109. 629
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tion, and ionic conditions lead to predictable changes in the relative migration rates of the various DNA conformational forms, thus defining conditions under which the separation and identification of complex mixtures can be accomplished. In the present investigation we have used agarose gel electrophoresis and autoradiography to analyze the DNA structures formed during the infection of Escherichia coli by bacteriophage pX174. We describe electrophoretic conditions and strategies for resolving all known intracellular viral and host DNA species and report the partial characterization of several novel forms of linear and circular molecules. The results are discussed in terms of the participation of these species as intermediates in pX174 replication, recombination, and transcription. MATERLALS AND METHODS Phage and bacterial strains. The lysis-defective amber mutant of bacteriophage 4pX174, am3, was used as a representative of the wild type with respect to viral DNA replication (22). E. coli C is the nonpermissive host strain for 4Xam3 (5). Medium and solutions. Low-phosphate TPG medium (24) was used for cell growth. Borate-EDTA consisted of 50 mM sodium borate-5 mM EDTA. TrisEDTA consisted of 50 mM Tris-hydrochloride-5 mM EDTA (pH 8.0). TEN buffer was 10 mM Tris-1 mM EDTA-10 mM NaCl (pH 7.5). Cyanide-azide consisted of 0.5 M potassium cyanide-0.03 M sodium azide. TAE buffer was 40 mM Tris base-5 mM sodium acetate-1 mM EDTA (pH 7.8). pH adjustments of buffers were made at room temperature, generally on 10-fold-concentrated stock solutions. Enzymes. Endonuclease R * PstI was purchased from Bethesda Research Labs (Bethesda, Md.). RNase H from calf thymus (33) was the gift of E. Chargaff and J. Stavrianopoulos (Roosevelt Hospital, New York, N.Y.). Preparation of DNA from infected cells. Phage, replicative form (RF), and viral DNA markers were labeled and purified as previously described (24). Cultures of E. coli C (100 ml) were grown to 4 x 108 cells per ml in TPG medium and infected with 32P-labeled 4X174 am3 at the multiplicity of infection indicated in the figure legends. Alternatively, cells were infected with unlabeled phage 30 min after the addition of 5 mCi of carrier-free H332P04 (Amersham Corp., Arlington Heights, Ill.). Samples (20 ml) were removed at various times during infection, mixed with 2 ml of cyanide-azide, and rapidly cooled. Cells were collected by centrifugation, and uneclipsed phage was removed by washing and recentrifugation five times with borate-EDTA (27). Cells were finally suspended in 1.75 ml of cold Tris-EDTA. Egg white lysozyme (Sigma Chemical Co., St. Louis, Mo.) was added to 275 ,ug/ml, and the solution was incubated on ice for 30 min. Sodium dodecyl sulfate was added to 1%, and incubation was continued at 37°C for 15 min. The solution was adjusted to 1 M NaCl and incubated at 0°C for approximately 5 h, followed by centrifugation to re-
move the resulting precipitate of high-molecularweight DNA (19). The supernatant was diluted 1:1 with Tris-EDTA and extracted with an equal volume of phenol equilibrated with 0.5 M Tris-5 mM EDTA (pH 8.0). DNA was precipitated from 0.3 M sodium acetate and 70% ethanol at -20°C and dissolved in 100 to 200 ll of TEN buffer. RF DNA was also prepared similarily from cells infected with phage in the presence of 30 jug of chloramphenicol per ml and purified by two neutral sucrose gradient centrifugations as described below. Agarose gel electrophoresis, denaturation, and densitometry. Electrophoretic analysis of 4X174 RF DNA was performed as previously described (24). Agarose slab gels (1.4%) were run at 5 V/cm for 13 h or 10 V/cm for 4 h in TAE buffer. Alkali-denatured samples were prepared by adding adequate 2 N NaOH to titrate the sample to a pH between 11.5 and 12.0, incubating at room temperature for 10 min, and reneutralizing with 1 M Tris-hydrochloride. Alternatively, DNA was denatured by heating to 90°C for 3 min, followed by rapid cooling on ice. Autoradiography of dried gels was performed using a Radelin SF-3 X-ray intensifying screen (General Electric Co., Schenectady, N. Y.) at -85°C. Films were quantitated with a model SL-504 Zeineh soft laser scanning densitometer (Biomed Instruments, Inc., Chicago, Ill.). The integrator feature of the densitometer was used at a film exposure time where the band integral was a linear function of DNA mass. Centrifugation analysis. Density gradients for sedimentation velocity analysis were linear 5 to 20% sucrose (Ultra-pure, Schwarz/Mann, Orangeburg, N.Y.) in Tris-EDTA containing 50 mM NaCl. Centrifugations were performed in a Spinco SW27 rotor at 10°C and 23,000 rpm for 20 h. A capillary tube was inserted through the gradient to 1 cm from the bottom, and 0.5-ml fractions were collected by pumping. This avoided contamination by the high-molecular-weight DNA which pellets to the bottom of the centrifuge tube. When necessary, samples were concentrated by alcohol precipitation before being applied to the gel. Electron microscopy. DNA was mounted for electron microscopy by the formamide method (11). Glyoxal fixation of DNA was by a procedure previously described (8).
RESULTS
Electrophoretic analysis of RF DNA fractionated by sucrose gradient velocity centrifugation. Figure 1 compares the resolution of agarose gel electrophoresis and sucrose gradient centrifugation for analysis of intracellular forms of bacteriophage OX174 DNA. Samples from representative fractions of the sucrose gradient were analyzed directly by agarose gel electrophoresis. Although centrifugation adequately separates the two major 4X replicative species (RFI, the supercoiled form which sediments at 21s, and RFII, the nicked circular duplex sedimenting at 16s), other RFs present in lower concentrations are not detected or resolved. The gel autoradiogram in Fig. 1 shows the following
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VOL. 32, 1979
variants. An electrophoretic analysis of DNA isolated from cells infected with labeled phage -.at various times in the absence of chloramphenicol is presented in Fig. 2. Samples were analyzed before and after controlled alkali denaturation, Tperformed under conditions adequate to dena- j ture species which are not covalently closed (+) duplex circles but not to alter the mobility of closed RF DNA (24). The relative levels of non1 , 1 B denatured RFs at various times indicate that .conversion of infecting viral DNA to the parental RF is complete within 5 min, and there is no 4 detectable processing of parental RF into single0 stranded circular DNA even after 40 min of infection. RFIII is present at approximately 1% of the total OX DNA throughout the infection a-) cycle. Several oligomeric forms can also be de1 i t tected but are present in significantly lower con2 , \Jf \ centrations. 1A We interpret the series of bands migrating X a\trt, () between the RFI and RFII positions as closed circular RF molecules of altered topological linking number, a, and assume that any two successive bands differ from each other by one superhelical turn (25, 31). These bands are covalently | 7'4 j
