vr~~x.oG~
72, 72-79
(1976)
On the Origin
of Bacteriophage 4X174 Replication
PETER Institute
of
J. WEISBEEK’
AND
Replicative
Form DNA
G. A. VAN ARKEL
Genetics and Institute of Molecular Biology, State University of Utrecht, Utrecht, The Netherlands Accepted February 16,1976
The origin of RF DNA replication of 4X174, as identified by the method of repair of heteroduplex DNA (Baas and Jansz, 19721, has been mapped more accurately, within gene A. Heteroduplexes of the wild-type minus strand and the plus (viral) strand of several well-localized gene A amber mutants were used to infect permissive spheroplasts. Single-burst experiments showed that the percentage of spheroplasts producing phages of both genotypes depended on the position on the genetic map of the mismatch in the heteroduplex DNA. A jump in the amount of repair of the heteroduplexes was found between the mutants am35 and amI%. Both mutants map in the far distal part of gene A at a distance of less than 200 nucleotides from gene B. The consequences of these results are discussed.
cued by coinfecting phages with a wildtype gene A (Baker and Tessman, 1967). This cis-activity may be explained by inactivation of the gene A protein after nicking of the DNA or by tight binding of the inactive mutant protein fragment to the DNA, thereby preventing wild-type protein molecules from acting. The replication of the RF DNA proceeds clockwise with regard to the genetic map and its origin has been found to be located in gene A by genetic analysis of spheroplasts that were infected with heteroduplex DNA (Baas and Jansz, 1972) and by labeling of actively replicating RF molecules (Godson, 1974). The experiments of Godson (1974) located the origin around the junction of the fragments R3 and R4 of the Hind II fragment map. From the combined genetic map fragment map presented in the accompanying paper (Weisbeek et al., 19761, it can be seen that this junction maps in that part of gene A that is proximal to gene H. The gap that has been found in RF11 during RF or ss DNA synthesis (Eisenberg and Denhardt, 1974; Godson, 1974; Johnson and Sinsheimer, 1974) is also found in R3. It seems attrac-
INTRODUCTION
The replication of the single-stranded DNA of bacteriophage +X174 includes three stages: the conversion of the infecting single-stranded DNA into a doublestranded parental RF (replicative form) molecule, the synthesis of progeny RF DNA and the production of singlestranded viral DNA (Sinsheimer, 1968). The first step is performed by host cell enzymes (Kornberg, 1974); it proceeds under conditions where synthesis of phagespecific proteins is inhibited (Tessman, 1966). The sequence of events has been worked out (Kornberg, 1974); whether the synthesis of the complementary strand starts at a specific point or is initiated randomly is as yet unsolved. The replication of the double-stranded parental RF DNA starts with the introduction of a nick in the viral (plus) strand by the phage gene A protein (Francke and Ray, 1972; Henry and Knippers, 1974). The gene A protein is cis-acting; conditionally lethal mutants in gene A cannot be res’ Author addressed.
to whom
requests
for reprints
should
be 72
Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
ORIGIN
OF
bX174
RF
tive to assume that these gaps are the consequences of the way the DNA synthesis is initiated, e.g., through RNA priming and subsequent removal of the RNA sequences. The synthesis of the single-stranded viral DNA is thought to proceed via a rolling circle model in which the plus strand is continuously peeled off from a DNA structure with a circular minus strand and a linear plus strand (Dressler, 1970). The linear strand is cut into molecules of genome length that are circularized and packed into .phage coats. RF11 molecules that are isolated during the ss DNA synthesis also contain a gap in the R3 fragment (Johnson and Sinsheimer, 1974). In this paper we describe an extension of the heteroduplex DNA experiments of Baas and Jansz (1972), with more gene A mutants that have been mapped accurately. The localization of the origin of RF DNA replication by heteroduplex mapping is based on the observations of Baas and Jansz (1972) that (i) after spheroplast infection a small fraction of the cells infected with heteroduplexes yielded phages of both the wild-type and the mutant genotype and that (ii) the size of this fraction of mixed bursts depended on the position of the mismatch on the genetic map. Combined with their assumption that the replication of double-stranded $X174 DNA starts at a fixed point (the origin) and that transcription, translation, and replication are not affected by the nature or position of a particular heteroduplex region, they concluded that there is competition between repair and replication. A heteroduplex region that is replicated early during the replication cycle is more likely to escape the repair process than a heteroduplex region that is located at a greater distance from the origin and that therefore is replicated later. Their experimental results showed a gradient of repair that starts low within gene A and increases clockwise around the genetic map. From this they determined the origin of RF DNA replication to be in gene A and the DNA synthesis to proceed clockwise on the genetic map. Comparable work has been done by
DNA
73
REPLICATION
Spatz and Trautner (1970) for the B. subtilis phage SPPl. Gene A, however, is a large gene; its size is about 1400 nucleotides and it occupies one-fourth of the genome. A more detailed localization of the origin of DNA replication is of great interest. Since the heteroduplex mapping depends on the known position of genetic markers, a detailed and reliable genetic map is a necessity. Since the genetic map derived from recombination analysis does not correspond to the physical map within the gene A region (Benbow et al., 1971; Hayashi and Hayashi, 1974) the construction of a genetic map by genetic analysis of restriction enzyme fragments of DNA (see preceding paper) makes it possible now to extend the origin mapping with heteroduplex DNA. Since we already knew that the origin is located in gene A, we concentrated upon gene A heteroduplexes. We constructed heteroduplexes from wild-type minus strand with plus strand DNA of five welllocalized gene A mutants and three mutants in other genes. The results show that the origin of RF DNA replication, as based on heteroduplex analysis, is located in the far distal part of gene A, less than 200 nucleotides from the boundary with gene B, a region not covered by the fragment R3 but by R5. MATERIALS
AND
METHODS
Bacteriophage mutants. $X174 wt is the wild-type phage (Sinsheimer, 1959). Am3(E), amlG(B), amlS(A), am35(A), am50(A), am3O(A), am86(A), and amS(G) are conditional lethal mutants, and are gifts of Dr. R. L. Sinsheimer. Bacterial strains. Escherichia coli C (BTCC 122) is the standard sup- host for $X174. It is used to grow and assay wildtype phage. E. coli HF4712 (sup&& is used to grow and assay the amber mutants (Benbow et aZ., 1974). E. coli K58 (sup& and UAA) is 4X-resistant and is used to make spheroplasts. It is an efficient host for the DNA of all mutants used (Zinder and Cooper, 1964). Media. 3XD broth, TKB broth, bottom agar and top agar are described by Borrias et al. (1969); PAM-medium by Guthrie and
74
WEISBEEK
AND
Sinsheimer (1963). 2 x SSC is 0.3 M NaCI0.03 M sodium citrate (pH 7.0). Phage growth and preparation of DNA. E. coli C in 3XD broth or HF4712 in TKB
broth were grown to 5 x 108 cells/ml and infected with phage at a multiplicity of 3. The cells were pelleted after 2 hr and lysozyme-treated. The debris was removed by centrifugation (12,000 g, 10 min) and the phage was pelleted (35,000 g, 120 min). The suspended phage was banded in a RbCl equilibrium density gradient (Jansz et al., 1966). The purified phage was phenol-extracted (Sinsheimer, 1959), and the DNA was further purified over a neutral sucrose gradient (5-20% sucrose in 0.3 M NaCl-0.05 M sodium citrate for 15 hr at 22,000 rpm in the Spinco SW25 rotor). RF DNA was prepared according to Jansz et al. (1966); the closed circular form (RFD was isolated from a CsCl gradient containing ethidium bromide (Radloff et al., 1967). RF1 was converted into RF11 by pancreatic deoxyribonuclease as described by Jansz et al. (1968). Isolation of the minus strand. The minus strand was obtained by heat denaturation for 3 min at 90” in 0.01 M Tris-0.001 M EDTA (pH 8.5) of double-stranded DNA (75 pg/ml) containing about equal quantities RF1 and RFII, in the presence of the same amount of poly(U,G). After cooling in an ice-bath, solid CsCl was added to a density of 1.73 g/ml. The mixture was centrifuged in polyallomer tubes in the Spinco 50 rotor (angle head) at 38,000 rpm for 70 hr at 10”. After the run fractions of 0.25 ml were collected and adjusted to 1.0 ml with distilled water, the absorbance was determined at 260 nm (Baas and Jansz, 1971). The purity of the minus strand was checked by incubation at 64” to allow annealing of contaminating plus strand material with the minus strands. The amount of resulting double-stranded DNA is a measure of the plus strand contamination and can be recorded by the differential uv inactivation of single- and double-stranded DNA (Sinsheimer et al., 1962) or can be visualized electronmicroscopically (KleinSchmidt et al., 1962). Preparation of heteroduplexes. One volume of wild-type minus strand (20 pg/ml)
VAN
ARKEL
was annealed to 2 vol of mutant plus (viral) strand (20 pg/ml) in 2 x SSC. This mixture was kept 2 min at 100” and then 60 min at 64”. After cooling, the DNA was filtered over an MF14 filter, which retains single-stranded DNA while doublestranded DNA passes through (Jansz et al ., 1966). The eluate contained the heteroduplex DNA which appeared under the electron microscope as full length doublestranded circular DNA. Preparation and plasts. Heteroduplex
infection
of sphero-
and single-stranded DNA was assayed on spheroplasts of E. coli K58. Spheroplasts were prepared according to Guthrie and Sinsheimer (1960), except for the use of PAM-medium instead of nutrient broth. For the single-burst experiments, equal volumes of spheroplasts and heteroduplex DNA (0.5 pug/ml in 0.05 M Tris, pH 7.0) were mixed and incubated for 6 min at 30”. The infected spheroplasts were then diluted with PAM-medium containing 2% BSA. From an appropriate dilution O.l-ml samples were added to 300 tubes containing 0.5 ml of PAM-medium. The tubes were incubated for 3 hr at 30”, thawed and frozen three times, and titrated. Ultraviolet irradiation. Ultraviolet irradiation was performed by illumination of DNA samples with a low-pressure mercury tube (Philips, 30 W, T.U.V.) at a distance of 55 cm. RESULTS
Preparation
AND
DISCUSSION
of Heteroduplexes
The two strands of denatured +X RF DNA bind different amounts of poly(U,G), due to their unequal A + C content (Sinsheimer, 1959; Opara-Kubinska et al., 1964). The minus strand (A + C = 56.9%) binds more poly(U,G) than the plus strand (A + C = 43.1%) and will therefore band at a higher buoyant density in a CsCl gradient. This is outlined in Fig. 1. The heavy ODpeak of the fractions 6 through 10 contains the minus strand, fractions 11 through 15 contain the viral plus strand, and the peak around fraction 26 is that portion of the RF1 that escaped nicking (Baas and Jansz, 1971). The minus strand fractions were pooled and analyzed for purity by means of
agreement with the results Jansz (1971).
FIG. 1. CsCl equilibrium density centrifugation of DNase-treated RF1 DNA, denatured in the presence of poly(U,G). The DNA was run for 70 hr at 38,000 rpm in the Spinco Type 50 rotor. Fractions of 0.25 ml were collected and adjusted to 1 ml with water..-0, Absorbance at 260 nm; 0- -0, phage titer in the spheroplast assay. The density increases from right to left. Fractions 6 through 10 represent the minus strand, 11 through 15 the plus strand, and 25, 26, 27 the native RF1 DNA, which bands at a buoyant density of 1.706 g/cm3.
uv inactivation and electron microscopy (see Materials and Methods). Both methods indicated 2-3% full-length doublestranded molecules, the rest was singlestranded DNA. This preparation of minus strand DNA was used to construct heteroduplexes. Heteroduplex DNA was prepared by annealing the wild-type minus strand with mutant plus (viral) strands and removing of single-stranded DNA (see Materials and Methods). Under the electron microscope the heteroduplexes appeared as fully double-stranded DNA circles; their uv inactivation is, however, intermediate between ss and ds DNA (see Fig. 2). This intermediate inactivation is most likely due to the presence of the mismatch and not to the occurrence of single-stranded regions, because annealing of the minus strand with wild-type plus strand (giving a homoduplex) results in DNA molecules that are inactivated exactly as RF DNA. This is in
of Baas and
Single-Burst Experiments Spheroplasts infected with heteroduplex DNA may yield any of three different kinds of burst: pure wild-type phage, pure mutant phage, or a mixture containing both wild-type and mutant phage. The majority of the infecting heteroduplexes is converted by repair enzymes into homoduplexes of either the wild type or the mutant DNA, and they therefore produce bursts of only one genotype. Spheroplasts were infected with the heteroduplex DNA and the progeny of single cells were analyzed (see Materials and Methods) to see whether one or both parental genotypes were present. The result of these analyses are given in Table 1. The last column of the table gives the percentages of infected cells that yield progeny of both phage types; this represents those infected cells in which the heteroduplex DNA has replicated before the mismatch was repaired. The amount of unrepaired heteroduplexes is highest for aml8(A) and decreases over amlG(B) and am3(E) until
t 20
40
60
80
0
FIG. 2. Ultraviolet survival of heteroduplex DNA of wild-type minus strand and am18 plus strand in the spheroplast assay (Cl-~-O), RF1 DNA (O--O), and viral ss DNA (O-•).
76
WEISBEEK
AND VAN ARKEL TABLE
SINGLE-BURBT
Heteroduplex
(+)
Strand aml8(A) amlG(B) am3CE)
Number of Mean num-1 bursts testec1 ber of in1Pcted spher(-) Strand oplasts per test tube
wt wt wt
amS(G)
wt
um86(A)
wt
am30(A)
wt
am33(A)
wt
um35(A)
wt
99
0.39
64
0.30 0.46 0.47 0.41
90 112 101 51 131 65 148
0.19
49 12 40 45
51 94 90
-
1 WITH HETERODUPLEX
Number of bursts with wt phage (- strand)
Number of bursts with am phage (+ strand)
[Aa Number of Percentage bursts with mixed bursts am and wt (Poisson corrected) phwe
;: = 66.2%
13 8 = 12.9%
22 12
= 20.9%
18.4
;; = 52.0%
25 32
20 20
= 19.8%
14.9
57 33
= 59.2%
25 11 = 23.7%
19 7 = 17.1%
12.5
101 52
= 78.1%
20
10 4
= 7.1%
3.1
= 64.5%
36 17
= 26.9%
13 4
= 8.6%
3.6
= 63.4%
22 12
= 30.4%
5 2
= 6.2%
2.7
14
19 = 32.4%
4 4
= 7.8%
3.9
29
12 6
= 9.8%
3.8
EXPERIMENTS
0.57 0.24 0.36 0.22 0.27 0.14 0.17 0.26 0.47 0.36
99 28 45 26 21 34 53 53
= 59.8% = 57.6%
I )h
= 28.2%
9 = 14.8%
31
= 32.6% -
(1Permissive spheroplasts were infected with heteroduplex DNA formed from wild-type minus strand and plus strand of different amber mutants. The infected spheroplasts were distributed over 300 tubes and incubated for 3 hr at 30”. The progeny phage, obtained after lysis of the spheroplasts, was analyzed with respect to the genetic marker.
it reaches an apparent minimum level in gene G (am91 that continues into gene A (a&6, am30, am50, and um35). The correlation of the amount of repair with the map positions of the mismatches is shown in Fig. 3, in which the percentages of mixed bursts are plotted against a linear genetic map. From Fig. 3 it follows that between the gene A mutants am35 and am18 there is a drastic change in the amount of repair of the heteroduplexes. Both am35 and am18 map to the extreme distal side of gene A (see Fig. 4). Assuming that the gradient in heteroduplex repair is directly related to the onset of DNA replication, i.e., that the change in amount of repair of heteroduplexes from one mutant to the other is caused by the origin lying between them, this means that the origin of RF DNA replication falls in gene A very close to gene B. Mutants um35(A), uml8(A), and umlG(B) all fall on a DNA stretch that measures 210 nucleotides and that is part of fragment R5 (see accompanying paper); moreover on the basis of available space on the genetic map, gene B should occupy at least half of this frag-
ment. For that reason am35 and am18 are most likely not more than 100 nucleotides away from the border between the genes A and B. In Table 1 the number of bursts that contain only wild-type phage always exceeds the number containing only mutant phage. This marked preference for repair of the heteroduplex towards the wild-type genotype is absent or even slightly reversed in the experiments of Baas and Jansz (1972). We have no unequivocal explanation for this effect. In contrast to the results reported here, recent experiments on 4X RF DNA replication (Godson, 1974) in which the terminus of replication was labeled, indicate the terminus and therefore also the origin, to be in the left half of gene A (at the R3/R4 split in the Hinal II fragment map). Also the gap in the viral strand of replicating RF DNA is placed in this region (Johnson and Sinsheimer, 1974; Godson, 1974; Eisenberg and Denhardt, 1974). If this gap has a role in the initiation of DNA synthesis, this result also points to an origin in the left part of gene A.
ORIGIN
OF
$X174
RF
DNA
77
REPLICATION
l\
f
.
. \
\
.
\
.
.
FIG. 3. The percentage heteroduplexes (see Table
of mixed l), plotted
.
bursts, obtained in single bursts of spheroplasts infected with against the linear genetic map as obtained in the accompanying
FIG. 4. The genetic map of $X174, derived by genetic map is discussed in detail in the preceding paper.
The part of the gene that falls between the heteroduplex origin and the origin as determined by Godson (1974), i.e., the region between am33 and am35, is exactly
analysis
of restriction
that part of gene have been found sence of known cate a structural
enzyme
DNA
different paper.
fragments.
The
A in which no mutations yet (see Fig. 4). This abmutants could well indiabnormality that might
78
WEISBEEK
AND VAN ARKEL
function somehow in the process of initiation of C#BXDNA synthesis. Work is in progress to characterize genetically this empty region. This may lead to an understanding of why the different methods applied in localizing the origin of DNA synthesis produce different results. ACKNOWLEDGMENTS The authors wish to thank Dr. W. Keegstra for the electron microscopical analyses, and Miss Anne To Wassenaar for her excellent technical assistance. REFERENCES BAAS, P. D., and JANSZ, H. S. (1971). duplexes: Preparation and some physical properties. Proc. Roy. Neth. 74, 191-206. BAAS, P. D., and JANSZ, H. S. (1972). tive form DNA replication, origin J. Mol.
Biol.
4X174 heterobiological and Acad.
Sci. C,
+X174 replicaand direction.
63, 569-576.
BAKER, R., and TESSMAN, I. (1967). The circular genetic map of phage S13. Proc. Nat. Acad. Sci. USA
58, 1438-1445.
BENBOW, R. M., HUTCHISON III, C. A., FABRICANT, J. D., and SINSHEIMER, R. L. (1971). Genetic map of bacteriophage 4x174. J. Virol. 7, 549-558. BENBOW, R. M., ZUCCARELLI, A. J., DAVIS, G. C., and SINSHEIMER, R. L. (1974). Genetic recombination in bacteriophage +X174. J. Virol. 13,898-907. BORRIAS, W. E., VAN DE POL, J. H., VAN DE VATE, C., and VAN ARKEL, G. A. (1969). Complementation experiments between conditional lethal mutants of bacteriophage 4X174. Molec. Gen. Genet. 105, 152-163. DRESSLER,D. (1970). The rolling circle for +X DNA replication, II. Synthesis of single-stranded circles. Proc. Nat. Acad. Sci. USA 67, 1934-1942. EISENBERG, S., and DENHARDT, D. T. (1974). The mechanism of replication of 4X174 singlestranded DNA. X. Distribution of the gaps in nascent +X174 replicative form DNA. Biochem. Biophys. Res. Commun. 61, 532-537. FRANCKE, B., and RAY, D. S. (1972). Cis-limited action of the gene A product of bacteriophage $X174 and the essential bacterial site. Proc. Nat. Acad. Sci. GODSON, G.
USA
69, 475-479.
N. (1974). Origin and direction of $X174 double- and single-stranded DNA synthesis. J. Mol. Biol. 90, 127-141. GUTHRIE, G. D., and SINSHEIMER, R. L. (1960). Infection of protoplasts of E. coli by subviral particles of bacteriophage 4x174. J. Mol. Biol. 2, 297305. GUTHRIE, G. D., and SINSHEIMER, R. L. (1963). Observations on the infections of bacterial proto-
plasm with Biochim.
the DNA of bacteriophage
Biophys.
Acta
4x174.
72, 296-297.
HAYASHI, M. N., and HAYASHI, M. (1974). Fragment maps of $X174 replicative form DNA produced by restriction enzymes from Haemophilus aphirophilus and H. influenzae H-I. J. Virol. 14, 1142-1151. HENRY, T. J., and KNIPPERS, R. (1974). Isolation and function of the gene A initiator of bacteriophage 4X174, a highly specific DNA endonuclease. Proc. Nat. Acad. Sci. USA 71, 1549-1553. JANSZ, H. S., POUWELS, P. H., and SCHIPHORST,J. (1966). Preparation of double-stranded DNA (replicative form) of bacteriophage 4X174: A simplified method. Biochim. Biophys. Acta 123, 626-627. JANSZ, H. S., BAAS, P. D., POUWELS, P. H., VAN BRUGGEN, E. F. J., and OLDENZIEL, H. (1968). Structure of the replicative form of bacteriophage 4X174. V. Interconversions between twisted, extended and randomly coiled forms of cyclic DNA. J. Mol. Biol. 32, 159-168. JOHNSON, P. H., and SINSHEIMER, R. L. (1974). Structure of an intermediate in the replication of bacteriophage 1$X174 deoxyribonucleic acid: The initiation site for DNA replication. J. Mol. Biol. 83, 47-61. KLEINSCHMIDT, A. K., LANG, D., JACHERTS, D., and ZAHN, R. V. (1962). Darstellung und Langenmessungen des gesamten Desoxyribonucleinsaure-Inhaltes von TZ-Bakteriophagen. Biochim. Biophys. Acta 61, 857-864. KORNBERG, A. (1974). “DNA Synthesis.” Freeman and Co., San Francisco. OPARA-KUBINSKA, Z., KUBINSKI, H., and SZYBALSKI, W. (1964). Interaction between denaturated DNA, polyribonucleotides and ribosomal RNA: Attempts at preparative separation of the complementary DNA strands. Proc. Nat. Acad. Sci. USA 52, 923-930. RADLOFF, R., BAUER, W., and VINOGRAD, J. (1967). A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in Hela cells. Proc. Nat. Acad. Sci. USA 57, 1514-1521. SINSHEIMER, R. L. (1959). Purification and properties of bacteriophage +X174. J. Mol. Biol. 1,37-42. SINSHEIMER, R. L. (1968). Bacteriophage +X174 and related viruses. Pragr. Nucl. Acid Res. Mol. Bial. 8, 115-169. SINSREIMER, R. L., STARMAN, B., NAGLER, C., and GUTHRIE, S. (1962). The process of infection with bacteriophage +X174. I. Evidence for a replicative form. J. Mol. Biol. 4, 142-160., SPATZ, H. CH., and TRAUTNER, T. A. (1970). One way to do experiments on gene conversion? Transfections with heteroduplex SPPl DNA. Molec. Gen. Genet. 109, 84-106. TESSMAN, E. S. (1966). Mutants ofbacteriophage S13 blocked in infectious DNA synthesis. J. Mol. Biol.
ORIGIN
OF
4X174
17, 218-236. WEISBEEK, P. J., VEREIJKEN, J. M., BAAS, P. D., JANSZ, H. S., and VAN ARKEL, G. A. (1976). The genetic map of bacteriophage $X174 constructed with restriction enzyme fragments. Virology 72,
RF
DNA
REPLICATION
79
61-71. ZINDER, N. D., and COOPER, S. (1964). Host dependent mutants of the bacteriophage f2. I. Isolation and preliminary classification. Virology 23, 152158.
72, 72-79
(1976)
On the Origin
of Bacteriophage 4X174 Replication
PETER Institute
of
J. WEISBEEK’
AND
Replicative
Form DNA
G. A. VAN ARKEL
Genetics and Institute of Molecular Biology, State University of Utrecht, Utrecht, The Netherlands Accepted February 16,1976
The origin of RF DNA replication of 4X174, as identified by the method of repair of heteroduplex DNA (Baas and Jansz, 19721, has been mapped more accurately, within gene A. Heteroduplexes of the wild-type minus strand and the plus (viral) strand of several well-localized gene A amber mutants were used to infect permissive spheroplasts. Single-burst experiments showed that the percentage of spheroplasts producing phages of both genotypes depended on the position on the genetic map of the mismatch in the heteroduplex DNA. A jump in the amount of repair of the heteroduplexes was found between the mutants am35 and amI%. Both mutants map in the far distal part of gene A at a distance of less than 200 nucleotides from gene B. The consequences of these results are discussed.
cued by coinfecting phages with a wildtype gene A (Baker and Tessman, 1967). This cis-activity may be explained by inactivation of the gene A protein after nicking of the DNA or by tight binding of the inactive mutant protein fragment to the DNA, thereby preventing wild-type protein molecules from acting. The replication of the RF DNA proceeds clockwise with regard to the genetic map and its origin has been found to be located in gene A by genetic analysis of spheroplasts that were infected with heteroduplex DNA (Baas and Jansz, 1972) and by labeling of actively replicating RF molecules (Godson, 1974). The experiments of Godson (1974) located the origin around the junction of the fragments R3 and R4 of the Hind II fragment map. From the combined genetic map fragment map presented in the accompanying paper (Weisbeek et al., 19761, it can be seen that this junction maps in that part of gene A that is proximal to gene H. The gap that has been found in RF11 during RF or ss DNA synthesis (Eisenberg and Denhardt, 1974; Godson, 1974; Johnson and Sinsheimer, 1974) is also found in R3. It seems attrac-
INTRODUCTION
The replication of the single-stranded DNA of bacteriophage +X174 includes three stages: the conversion of the infecting single-stranded DNA into a doublestranded parental RF (replicative form) molecule, the synthesis of progeny RF DNA and the production of singlestranded viral DNA (Sinsheimer, 1968). The first step is performed by host cell enzymes (Kornberg, 1974); it proceeds under conditions where synthesis of phagespecific proteins is inhibited (Tessman, 1966). The sequence of events has been worked out (Kornberg, 1974); whether the synthesis of the complementary strand starts at a specific point or is initiated randomly is as yet unsolved. The replication of the double-stranded parental RF DNA starts with the introduction of a nick in the viral (plus) strand by the phage gene A protein (Francke and Ray, 1972; Henry and Knippers, 1974). The gene A protein is cis-acting; conditionally lethal mutants in gene A cannot be res’ Author addressed.
to whom
requests
for reprints
should
be 72
Copyright All rights
8 1976 by Academic Press, Inc. of reproduction in any form reserved.
ORIGIN
OF
bX174
RF
tive to assume that these gaps are the consequences of the way the DNA synthesis is initiated, e.g., through RNA priming and subsequent removal of the RNA sequences. The synthesis of the single-stranded viral DNA is thought to proceed via a rolling circle model in which the plus strand is continuously peeled off from a DNA structure with a circular minus strand and a linear plus strand (Dressler, 1970). The linear strand is cut into molecules of genome length that are circularized and packed into .phage coats. RF11 molecules that are isolated during the ss DNA synthesis also contain a gap in the R3 fragment (Johnson and Sinsheimer, 1974). In this paper we describe an extension of the heteroduplex DNA experiments of Baas and Jansz (1972), with more gene A mutants that have been mapped accurately. The localization of the origin of RF DNA replication by heteroduplex mapping is based on the observations of Baas and Jansz (1972) that (i) after spheroplast infection a small fraction of the cells infected with heteroduplexes yielded phages of both the wild-type and the mutant genotype and that (ii) the size of this fraction of mixed bursts depended on the position of the mismatch on the genetic map. Combined with their assumption that the replication of double-stranded $X174 DNA starts at a fixed point (the origin) and that transcription, translation, and replication are not affected by the nature or position of a particular heteroduplex region, they concluded that there is competition between repair and replication. A heteroduplex region that is replicated early during the replication cycle is more likely to escape the repair process than a heteroduplex region that is located at a greater distance from the origin and that therefore is replicated later. Their experimental results showed a gradient of repair that starts low within gene A and increases clockwise around the genetic map. From this they determined the origin of RF DNA replication to be in gene A and the DNA synthesis to proceed clockwise on the genetic map. Comparable work has been done by
DNA
73
REPLICATION
Spatz and Trautner (1970) for the B. subtilis phage SPPl. Gene A, however, is a large gene; its size is about 1400 nucleotides and it occupies one-fourth of the genome. A more detailed localization of the origin of DNA replication is of great interest. Since the heteroduplex mapping depends on the known position of genetic markers, a detailed and reliable genetic map is a necessity. Since the genetic map derived from recombination analysis does not correspond to the physical map within the gene A region (Benbow et al., 1971; Hayashi and Hayashi, 1974) the construction of a genetic map by genetic analysis of restriction enzyme fragments of DNA (see preceding paper) makes it possible now to extend the origin mapping with heteroduplex DNA. Since we already knew that the origin is located in gene A, we concentrated upon gene A heteroduplexes. We constructed heteroduplexes from wild-type minus strand with plus strand DNA of five welllocalized gene A mutants and three mutants in other genes. The results show that the origin of RF DNA replication, as based on heteroduplex analysis, is located in the far distal part of gene A, less than 200 nucleotides from the boundary with gene B, a region not covered by the fragment R3 but by R5. MATERIALS
AND
METHODS
Bacteriophage mutants. $X174 wt is the wild-type phage (Sinsheimer, 1959). Am3(E), amlG(B), amlS(A), am35(A), am50(A), am3O(A), am86(A), and amS(G) are conditional lethal mutants, and are gifts of Dr. R. L. Sinsheimer. Bacterial strains. Escherichia coli C (BTCC 122) is the standard sup- host for $X174. It is used to grow and assay wildtype phage. E. coli HF4712 (sup&& is used to grow and assay the amber mutants (Benbow et aZ., 1974). E. coli K58 (sup& and UAA) is 4X-resistant and is used to make spheroplasts. It is an efficient host for the DNA of all mutants used (Zinder and Cooper, 1964). Media. 3XD broth, TKB broth, bottom agar and top agar are described by Borrias et al. (1969); PAM-medium by Guthrie and
74
WEISBEEK
AND
Sinsheimer (1963). 2 x SSC is 0.3 M NaCI0.03 M sodium citrate (pH 7.0). Phage growth and preparation of DNA. E. coli C in 3XD broth or HF4712 in TKB
broth were grown to 5 x 108 cells/ml and infected with phage at a multiplicity of 3. The cells were pelleted after 2 hr and lysozyme-treated. The debris was removed by centrifugation (12,000 g, 10 min) and the phage was pelleted (35,000 g, 120 min). The suspended phage was banded in a RbCl equilibrium density gradient (Jansz et al., 1966). The purified phage was phenol-extracted (Sinsheimer, 1959), and the DNA was further purified over a neutral sucrose gradient (5-20% sucrose in 0.3 M NaCl-0.05 M sodium citrate for 15 hr at 22,000 rpm in the Spinco SW25 rotor). RF DNA was prepared according to Jansz et al. (1966); the closed circular form (RFD was isolated from a CsCl gradient containing ethidium bromide (Radloff et al., 1967). RF1 was converted into RF11 by pancreatic deoxyribonuclease as described by Jansz et al. (1968). Isolation of the minus strand. The minus strand was obtained by heat denaturation for 3 min at 90” in 0.01 M Tris-0.001 M EDTA (pH 8.5) of double-stranded DNA (75 pg/ml) containing about equal quantities RF1 and RFII, in the presence of the same amount of poly(U,G). After cooling in an ice-bath, solid CsCl was added to a density of 1.73 g/ml. The mixture was centrifuged in polyallomer tubes in the Spinco 50 rotor (angle head) at 38,000 rpm for 70 hr at 10”. After the run fractions of 0.25 ml were collected and adjusted to 1.0 ml with distilled water, the absorbance was determined at 260 nm (Baas and Jansz, 1971). The purity of the minus strand was checked by incubation at 64” to allow annealing of contaminating plus strand material with the minus strands. The amount of resulting double-stranded DNA is a measure of the plus strand contamination and can be recorded by the differential uv inactivation of single- and double-stranded DNA (Sinsheimer et al., 1962) or can be visualized electronmicroscopically (KleinSchmidt et al., 1962). Preparation of heteroduplexes. One volume of wild-type minus strand (20 pg/ml)
VAN
ARKEL
was annealed to 2 vol of mutant plus (viral) strand (20 pg/ml) in 2 x SSC. This mixture was kept 2 min at 100” and then 60 min at 64”. After cooling, the DNA was filtered over an MF14 filter, which retains single-stranded DNA while doublestranded DNA passes through (Jansz et al ., 1966). The eluate contained the heteroduplex DNA which appeared under the electron microscope as full length doublestranded circular DNA. Preparation and plasts. Heteroduplex
infection
of sphero-
and single-stranded DNA was assayed on spheroplasts of E. coli K58. Spheroplasts were prepared according to Guthrie and Sinsheimer (1960), except for the use of PAM-medium instead of nutrient broth. For the single-burst experiments, equal volumes of spheroplasts and heteroduplex DNA (0.5 pug/ml in 0.05 M Tris, pH 7.0) were mixed and incubated for 6 min at 30”. The infected spheroplasts were then diluted with PAM-medium containing 2% BSA. From an appropriate dilution O.l-ml samples were added to 300 tubes containing 0.5 ml of PAM-medium. The tubes were incubated for 3 hr at 30”, thawed and frozen three times, and titrated. Ultraviolet irradiation. Ultraviolet irradiation was performed by illumination of DNA samples with a low-pressure mercury tube (Philips, 30 W, T.U.V.) at a distance of 55 cm. RESULTS
Preparation
AND
DISCUSSION
of Heteroduplexes
The two strands of denatured +X RF DNA bind different amounts of poly(U,G), due to their unequal A + C content (Sinsheimer, 1959; Opara-Kubinska et al., 1964). The minus strand (A + C = 56.9%) binds more poly(U,G) than the plus strand (A + C = 43.1%) and will therefore band at a higher buoyant density in a CsCl gradient. This is outlined in Fig. 1. The heavy ODpeak of the fractions 6 through 10 contains the minus strand, fractions 11 through 15 contain the viral plus strand, and the peak around fraction 26 is that portion of the RF1 that escaped nicking (Baas and Jansz, 1971). The minus strand fractions were pooled and analyzed for purity by means of
agreement with the results Jansz (1971).
FIG. 1. CsCl equilibrium density centrifugation of DNase-treated RF1 DNA, denatured in the presence of poly(U,G). The DNA was run for 70 hr at 38,000 rpm in the Spinco Type 50 rotor. Fractions of 0.25 ml were collected and adjusted to 1 ml with water..-0, Absorbance at 260 nm; 0- -0, phage titer in the spheroplast assay. The density increases from right to left. Fractions 6 through 10 represent the minus strand, 11 through 15 the plus strand, and 25, 26, 27 the native RF1 DNA, which bands at a buoyant density of 1.706 g/cm3.
uv inactivation and electron microscopy (see Materials and Methods). Both methods indicated 2-3% full-length doublestranded molecules, the rest was singlestranded DNA. This preparation of minus strand DNA was used to construct heteroduplexes. Heteroduplex DNA was prepared by annealing the wild-type minus strand with mutant plus (viral) strands and removing of single-stranded DNA (see Materials and Methods). Under the electron microscope the heteroduplexes appeared as fully double-stranded DNA circles; their uv inactivation is, however, intermediate between ss and ds DNA (see Fig. 2). This intermediate inactivation is most likely due to the presence of the mismatch and not to the occurrence of single-stranded regions, because annealing of the minus strand with wild-type plus strand (giving a homoduplex) results in DNA molecules that are inactivated exactly as RF DNA. This is in
of Baas and
Single-Burst Experiments Spheroplasts infected with heteroduplex DNA may yield any of three different kinds of burst: pure wild-type phage, pure mutant phage, or a mixture containing both wild-type and mutant phage. The majority of the infecting heteroduplexes is converted by repair enzymes into homoduplexes of either the wild type or the mutant DNA, and they therefore produce bursts of only one genotype. Spheroplasts were infected with the heteroduplex DNA and the progeny of single cells were analyzed (see Materials and Methods) to see whether one or both parental genotypes were present. The result of these analyses are given in Table 1. The last column of the table gives the percentages of infected cells that yield progeny of both phage types; this represents those infected cells in which the heteroduplex DNA has replicated before the mismatch was repaired. The amount of unrepaired heteroduplexes is highest for aml8(A) and decreases over amlG(B) and am3(E) until
t 20
40
60
80
0
FIG. 2. Ultraviolet survival of heteroduplex DNA of wild-type minus strand and am18 plus strand in the spheroplast assay (Cl-~-O), RF1 DNA (O--O), and viral ss DNA (O-•).
76
WEISBEEK
AND VAN ARKEL TABLE
SINGLE-BURBT
Heteroduplex
(+)
Strand aml8(A) amlG(B) am3CE)
Number of Mean num-1 bursts testec1 ber of in1Pcted spher(-) Strand oplasts per test tube
wt wt wt
amS(G)
wt
um86(A)
wt
am30(A)
wt
am33(A)
wt
um35(A)
wt
99
0.39
64
0.30 0.46 0.47 0.41
90 112 101 51 131 65 148
0.19
49 12 40 45
51 94 90
-
1 WITH HETERODUPLEX
Number of bursts with wt phage (- strand)
Number of bursts with am phage (+ strand)
[Aa Number of Percentage bursts with mixed bursts am and wt (Poisson corrected) phwe
;: = 66.2%
13 8 = 12.9%
22 12
= 20.9%
18.4
;; = 52.0%
25 32
20 20
= 19.8%
14.9
57 33
= 59.2%
25 11 = 23.7%
19 7 = 17.1%
12.5
101 52
= 78.1%
20
10 4
= 7.1%
3.1
= 64.5%
36 17
= 26.9%
13 4
= 8.6%
3.6
= 63.4%
22 12
= 30.4%
5 2
= 6.2%
2.7
14
19 = 32.4%
4 4
= 7.8%
3.9
29
12 6
= 9.8%
3.8
EXPERIMENTS
0.57 0.24 0.36 0.22 0.27 0.14 0.17 0.26 0.47 0.36
99 28 45 26 21 34 53 53
= 59.8% = 57.6%
I )h
= 28.2%
9 = 14.8%
31
= 32.6% -
(1Permissive spheroplasts were infected with heteroduplex DNA formed from wild-type minus strand and plus strand of different amber mutants. The infected spheroplasts were distributed over 300 tubes and incubated for 3 hr at 30”. The progeny phage, obtained after lysis of the spheroplasts, was analyzed with respect to the genetic marker.
it reaches an apparent minimum level in gene G (am91 that continues into gene A (a&6, am30, am50, and um35). The correlation of the amount of repair with the map positions of the mismatches is shown in Fig. 3, in which the percentages of mixed bursts are plotted against a linear genetic map. From Fig. 3 it follows that between the gene A mutants am35 and am18 there is a drastic change in the amount of repair of the heteroduplexes. Both am35 and am18 map to the extreme distal side of gene A (see Fig. 4). Assuming that the gradient in heteroduplex repair is directly related to the onset of DNA replication, i.e., that the change in amount of repair of heteroduplexes from one mutant to the other is caused by the origin lying between them, this means that the origin of RF DNA replication falls in gene A very close to gene B. Mutants um35(A), uml8(A), and umlG(B) all fall on a DNA stretch that measures 210 nucleotides and that is part of fragment R5 (see accompanying paper); moreover on the basis of available space on the genetic map, gene B should occupy at least half of this frag-
ment. For that reason am35 and am18 are most likely not more than 100 nucleotides away from the border between the genes A and B. In Table 1 the number of bursts that contain only wild-type phage always exceeds the number containing only mutant phage. This marked preference for repair of the heteroduplex towards the wild-type genotype is absent or even slightly reversed in the experiments of Baas and Jansz (1972). We have no unequivocal explanation for this effect. In contrast to the results reported here, recent experiments on 4X RF DNA replication (Godson, 1974) in which the terminus of replication was labeled, indicate the terminus and therefore also the origin, to be in the left half of gene A (at the R3/R4 split in the Hinal II fragment map). Also the gap in the viral strand of replicating RF DNA is placed in this region (Johnson and Sinsheimer, 1974; Godson, 1974; Eisenberg and Denhardt, 1974). If this gap has a role in the initiation of DNA synthesis, this result also points to an origin in the left part of gene A.
ORIGIN
OF
$X174
RF
DNA
77
REPLICATION
l\
f
.
. \
\
.
\
.
.
FIG. 3. The percentage heteroduplexes (see Table
of mixed l), plotted
.
bursts, obtained in single bursts of spheroplasts infected with against the linear genetic map as obtained in the accompanying
FIG. 4. The genetic map of $X174, derived by genetic map is discussed in detail in the preceding paper.
The part of the gene that falls between the heteroduplex origin and the origin as determined by Godson (1974), i.e., the region between am33 and am35, is exactly
analysis
of restriction
that part of gene have been found sence of known cate a structural
enzyme
DNA
different paper.
fragments.
The
A in which no mutations yet (see Fig. 4). This abmutants could well indiabnormality that might
78
WEISBEEK
AND VAN ARKEL
function somehow in the process of initiation of C#BXDNA synthesis. Work is in progress to characterize genetically this empty region. This may lead to an understanding of why the different methods applied in localizing the origin of DNA synthesis produce different results. ACKNOWLEDGMENTS The authors wish to thank Dr. W. Keegstra for the electron microscopical analyses, and Miss Anne To Wassenaar for her excellent technical assistance. REFERENCES BAAS, P. D., and JANSZ, H. S. (1971). duplexes: Preparation and some physical properties. Proc. Roy. Neth. 74, 191-206. BAAS, P. D., and JANSZ, H. S. (1972). tive form DNA replication, origin J. Mol.
Biol.
4X174 heterobiological and Acad.
Sci. C,
+X174 replicaand direction.
63, 569-576.
BAKER, R., and TESSMAN, I. (1967). The circular genetic map of phage S13. Proc. Nat. Acad. Sci. USA
58, 1438-1445.
BENBOW, R. M., HUTCHISON III, C. A., FABRICANT, J. D., and SINSHEIMER, R. L. (1971). Genetic map of bacteriophage 4x174. J. Virol. 7, 549-558. BENBOW, R. M., ZUCCARELLI, A. J., DAVIS, G. C., and SINSHEIMER, R. L. (1974). Genetic recombination in bacteriophage +X174. J. Virol. 13,898-907. BORRIAS, W. E., VAN DE POL, J. H., VAN DE VATE, C., and VAN ARKEL, G. A. (1969). Complementation experiments between conditional lethal mutants of bacteriophage 4X174. Molec. Gen. Genet. 105, 152-163. DRESSLER,D. (1970). The rolling circle for +X DNA replication, II. Synthesis of single-stranded circles. Proc. Nat. Acad. Sci. USA 67, 1934-1942. EISENBERG, S., and DENHARDT, D. T. (1974). The mechanism of replication of 4X174 singlestranded DNA. X. Distribution of the gaps in nascent +X174 replicative form DNA. Biochem. Biophys. Res. Commun. 61, 532-537. FRANCKE, B., and RAY, D. S. (1972). Cis-limited action of the gene A product of bacteriophage $X174 and the essential bacterial site. Proc. Nat. Acad. Sci. GODSON, G.
USA
69, 475-479.
N. (1974). Origin and direction of $X174 double- and single-stranded DNA synthesis. J. Mol. Biol. 90, 127-141. GUTHRIE, G. D., and SINSHEIMER, R. L. (1960). Infection of protoplasts of E. coli by subviral particles of bacteriophage 4x174. J. Mol. Biol. 2, 297305. GUTHRIE, G. D., and SINSHEIMER, R. L. (1963). Observations on the infections of bacterial proto-
plasm with Biochim.
the DNA of bacteriophage
Biophys.
Acta
4x174.
72, 296-297.
HAYASHI, M. N., and HAYASHI, M. (1974). Fragment maps of $X174 replicative form DNA produced by restriction enzymes from Haemophilus aphirophilus and H. influenzae H-I. J. Virol. 14, 1142-1151. HENRY, T. J., and KNIPPERS, R. (1974). Isolation and function of the gene A initiator of bacteriophage 4X174, a highly specific DNA endonuclease. Proc. Nat. Acad. Sci. USA 71, 1549-1553. JANSZ, H. S., POUWELS, P. H., and SCHIPHORST,J. (1966). Preparation of double-stranded DNA (replicative form) of bacteriophage 4X174: A simplified method. Biochim. Biophys. Acta 123, 626-627. JANSZ, H. S., BAAS, P. D., POUWELS, P. H., VAN BRUGGEN, E. F. J., and OLDENZIEL, H. (1968). Structure of the replicative form of bacteriophage 4X174. V. Interconversions between twisted, extended and randomly coiled forms of cyclic DNA. J. Mol. Biol. 32, 159-168. JOHNSON, P. H., and SINSHEIMER, R. L. (1974). Structure of an intermediate in the replication of bacteriophage 1$X174 deoxyribonucleic acid: The initiation site for DNA replication. J. Mol. Biol. 83, 47-61. KLEINSCHMIDT, A. K., LANG, D., JACHERTS, D., and ZAHN, R. V. (1962). Darstellung und Langenmessungen des gesamten Desoxyribonucleinsaure-Inhaltes von TZ-Bakteriophagen. Biochim. Biophys. Acta 61, 857-864. KORNBERG, A. (1974). “DNA Synthesis.” Freeman and Co., San Francisco. OPARA-KUBINSKA, Z., KUBINSKI, H., and SZYBALSKI, W. (1964). Interaction between denaturated DNA, polyribonucleotides and ribosomal RNA: Attempts at preparative separation of the complementary DNA strands. Proc. Nat. Acad. Sci. USA 52, 923-930. RADLOFF, R., BAUER, W., and VINOGRAD, J. (1967). A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in Hela cells. Proc. Nat. Acad. Sci. USA 57, 1514-1521. SINSHEIMER, R. L. (1959). Purification and properties of bacteriophage +X174. J. Mol. Biol. 1,37-42. SINSHEIMER, R. L. (1968). Bacteriophage +X174 and related viruses. Pragr. Nucl. Acid Res. Mol. Bial. 8, 115-169. SINSREIMER, R. L., STARMAN, B., NAGLER, C., and GUTHRIE, S. (1962). The process of infection with bacteriophage +X174. I. Evidence for a replicative form. J. Mol. Biol. 4, 142-160., SPATZ, H. CH., and TRAUTNER, T. A. (1970). One way to do experiments on gene conversion? Transfections with heteroduplex SPPl DNA. Molec. Gen. Genet. 109, 84-106. TESSMAN, E. S. (1966). Mutants ofbacteriophage S13 blocked in infectious DNA synthesis. J. Mol. Biol.
ORIGIN
OF
4X174
17, 218-236. WEISBEEK, P. J., VEREIJKEN, J. M., BAAS, P. D., JANSZ, H. S., and VAN ARKEL, G. A. (1976). The genetic map of bacteriophage $X174 constructed with restriction enzyme fragments. Virology 72,
RF
DNA
REPLICATION
79
61-71. ZINDER, N. D., and COOPER, S. (1964). Host dependent mutants of the bacteriophage f2. I. Isolation and preliminary classification. Virology 23, 152158.
