Vol. 32, No. 2
JOURNAL OF VIROLOGY, Nov. 1979, p. 606-613
0022-538X/79/11-0606/08$02.00/0
Electron Microscopic Analysis of Partially Replicated Bacteriophage T7 DNA KATHY BAUMAN BURCK,1 DOUGLAS G. SCRABA,2 AND ROBERT C. MILLER, JR.`* Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1 W5,1 and Department of Biochemistry, University ofAlberta, Edmonton, Alberta, Canada2 Received for publication 30 May 1979
Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated molecules. The results are in excellent agreement with those of hybridization experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144-6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460-476, 1961; Doermann et al., J. Cell. Comp. Physiol. 45[Suppl.]:51-74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome.
Cross-reactivation refers to a process whereby genetic markers are rescued from a UV-irradiated, wild-type phage by a coinfecting mutant phage. The probability of a specific marker being rescued during T4 or T7 phage infection depends on the map position of the marker (3, 23). During T7 infection, only markers toward the left end of the molecules are rescued efficiently. Evidence has been presented which indicates that those markers which are rescued efficiently are markers which replicate efficiently (3); i.e., 32P_ labeled progeny DNA synthesized after infection by a UV-irradiated T7+ phage hybridizes predominantly with restriction fragments of T7+ DNA known to carry markers rescued efficiently during cross-reactivation. The effect is dose dependent: the higher the dose of UV irradiation, the fewer the markers which are rescued efficiently and the smaller the area of the genome which replicates efficiently. One idea which correlates the efficiency of replication with the efficiency of marker rescue is the partial-replica hypothesis (1, 6). This theory postulates that replication of a UV-irradiated genome starts from a specific origin(s) and proceeds in a bidirectional manner to UV lesions
which block further replication. Subsequently, those regions of the genome which have replicated most efficiently are rescued most efficiently by a coinfecting phage. The hybridization data mentioned above are in agreement with this hypothesis, the data clearly indicating the presence of partial replicas of UV-irradiated genomes after infection. This report presents the results of an electron microscopic analysis of partially replicated, UVirradiated T7+ DNA. It describes the distribution of replicated regions, growing forks, and bubbles in replicating, UV-irradiated T7+ DNA isolated from infected cells. In addition, a photoreactivation experiment is reported here which supports the hypothesis that thymine dimers normally block cross-reactivation in the right end of the T7 map. All of the results agree with and extend our previous conclusions and strongly support the partial-replica hypothesis. MATERIALS AND METHODS
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Bacterial and phage strains. Escherichia coli B23 (sup°) was used as the nonpermissive host for cross-reactivation experiments. After adaptation for growth in density medium (see below), B23 was used as the host for the isolation of partially replicated T7+
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DNA for electron microscopy. E. coli 011' (supE) was used as the permissive host during cross-reactivation experiments. All phage strains originally were provided by F. W. Studier. Our T7+ (wild-type) strain was determined to be free of deletions and like the original Studier strain by restriction endonuclease (HpaI and MboI) analysis (12). Namx is the general designation for amber mutants, where N is the gene number, and x is the specific mutant. The mutants used in this study were: lam193, lam323, lam342a, 2am64, 3am29, 4am208, 5am28, 6am233, 8amll, 11am37, 14am140, 16am194, 17am290, and l9amlO (20). Chemicals and isotopes. Thymidine was purchased from Worthington Biochemicals Corp. Uracil, 5-fluorodeoxyuridine, and cytochrome c (type V) were from Sigma Chemical Co. The density isotopes 2H20, '5NH4Cl, and deuterated algal whole hydrolysate were from Merck Sharp & Dohme. [methyl-3H]thymidine and 32P as H332PO4 were purchased from New England Nuclear Corp. Formamide was from Matheson, Coleman and Bell and was deionized before use by treatment with Bio-Rad AG 501-X8 mixed-bed resin. Media and buffers. Density medium contained the following (per liter of 2H20): 7.0 g of Na2HPO4, 3.0 g of KH2PO4, 1.0 g of '5NH4Cl, 0.5 g of NaCl, and 0.03 ml of 0.1 M FeCli. After autoclaving, the following were added: 1.0 ml of 1 M MgSO4, 0.1 ml of 1 M CaCl2, 12.5 ml of 20% glucose in 2H20, and 1.12 ml of deuterated algal whole hydrolysate (22). Tris-NaCl-EDTA buffer (TNE) contained 0.01 M Tris-hydrochloride, 0.15 M NaCl, and 0.015 M EDTA (pH 7.4). Tris-EDTA buffer contained 0.5 M Trishydrochloride and 0.05 M EDTA (pH 7.0). Lysis buffer contained 0.1 M NaCl, 0.02 M EDTA, 0.01 M KCN, 0.01 M iodoacetate, and 0.1 M Tris (pH 7.4) (22). T7 Tris salt was 1 M NaCl and 0.05 M Tris-hydrochloride
to be 12.0 + 0.5 [Lm in these experiments). Isolation of partially replicated molecules after infection of E. coli B23 by UV-irradiated T7+ phage. E. coli B23 was adapted for growth in the density medium by serial passage through 20, 40, 60, 75, 90, 95, and 100% substituted medium (22). The adapted cells had a generation time of 75 to 80 min at 37°C. Non-irradiated T7+ phage followed a normal single-step growth curve at 30°C in the density-labeled cells, giving a burst of 120 phage per cell by 45 min after infection. A 50-ml culture of density-labeled E. coli B23 was grown to approximately 3 x 108 cells per ml at 37°C in heavy medium containing 5 jig of thymidine per ml, 5, g of 5-fluorodeoxyuridine per ml, 25 ,ug of uracil per ml, and 500 ,uCi of [methyl-'H]thymidine and then shifted to 30°C for 15 min. This procedure allowed labeling of the bacterial cells for HH reference (both strands labeled) in CsCl gradients. The cells then were infected at a multiplicity of infection of 2 with UV-irradiated, 32P-labeled LL (no density label in either strand) T7+ containing 7.3 lethal events per genome. The 32p label was at a specific activity of 2 mCi/mg, which leads to the incorporation of a 32p atom in two-fifths of the phage particles. The multiplicity of infection was determined by monitoring surviving bacteria. Since no attempt was made in this experiment to overcome superinfection exclusion (2), a calculated multiplicity of infection of 2 does not necessarily mean that any cells received injected DNA from more than one phage. Infective centers and background phage also were monitored. UV-irradiated T7 phage are unable to conduct a productive infection: infective centers are typically only 0.1 to 0.2% of the control bacteria. Non-irradiated T7+ produce infective centers on 90% of the control bacteria. At 25 nmin postinfection, the culture was chilled by pipetting it into 2 volumes of ice-cold TNE plus 1 volume of lysis buffer. The infected cells were sedimented and resuspended at a concentration of 1.5 x 109 bacteria per ml in lysis buffer. Cells then were lysed with lysozyme (400,ug/ml, 0°C, 45 min) followed by detergent (0.1% sodium lauryl sarcosinate, 65°C, 20 min). The lysate was deproteinized by treatment with self-digested pronase (1 mg/ml, 37°C, 12 h). Samples of the lysate were mixed 1:4 with saturated CsCl (in distilled water) and centrifuged in an SB 283 rotor in a B60 International centrifuge at 30,000 rpm for 72 h at 12°C. Approximately 70 fractions were collected from the bottom of the tube and assayed for ;'H (HH E. coli DNA) and 32p (T7 DNA). Fractions banding on the heavy side of the T7 (P2P) peak were pooled and centrifuged in a Beckman type 65 rotor at 32,000 rpm for 72 h at 12°C. Specific fractions from the second gradient were
(pH 7.4).
UV irradiation of phage. T7+ phage were diluted
to 10" bacteriophage per ml in T7 Tris salt and placed in a plastic petri dish on ice. Irradiation was for 20 s at a distance of 30 cm from a General Electric G14T8 15W germicidal lamp. At this dose, T7 phage received approximately 7.3 lethal hits per particle. Lethal events were quantitated by plotting a survival curve
of the irradiated phage. Cross-reactivation and photoreactivation. Cross-reactivation experiments were performed as previously described (3). When photoreactivation was desired, cross-reactivation was performed under fluorescent-light illumination, and infective centers were incubated beneath General Electric F40 fluorescent lights for 15 h at room temperature. Electron microscopy. Selected fractions from CsCl gradients were dialyzed overnight (4°C) against 0.1 M Tris-10 mM EDTA (pH 7.0) before being mounted for electron microscopic examination by the 40%-10% formamide spreading procedure of Davis et al. (4). Partially replicated molecules were photographed at a magnification of x44,000 with a Phillips EM300 electron microscope operated at 60 kV and with a 30-nm-objective aperture. Molecular lengths were determined with a map measuring device from tracings of prints enlarged three times. All measurements were normalized to unit length T7 DNA (found
pooled for electron microscopy. Before centrifugation, the polyallomer tubes were treated for 1 h with 10% bovine serum albumin. Other methods. Preparation of '32P-labeled T7 bacteriophage and determination of label uptake into acid-insoluble material have been described previously
(13).
RESULTS Isolation of partially replicated T7+ DNA. Partially replicated T7 DNA molecules were
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isolated from density-labeled E. coli infected with UV-irradiated phage as described above. The DNA was sedimented to equilibrium in CsCl density gradients, and the distribution of radioactivity was determined. As expected, T7+ irradiated to 7.3 phage lethal events did not undergo even one round of replication as no parental DNA banded at the hybrid location. Material banding on the heavy side of the T7 DNA peak was pooled and resedimented in a second CsCl gradient (Fig. 1). The fractions of this gradient indicated by brackets were pooled and analyzed by electron microscopy. This figure shows that the T7 DNA was well separated from contaminating E. coli DNA. Electron microscopy of partially replicated T7+ DNA. Partially replicated, UV-irradiated T7+ DNA isolated from a CsCl gradient was examined by electron microscopy. Four categories of partially replicated molecules of T7 size DNA were distinguished: (i) eye forms, (ii) molecules containing forks with two equal arms, (iii) mrolecules containing both eye forms and forks, and (iv) molecules containing forks with arms of unequal length, where two of the branches together were of T7 length; class iv molecules were assumed to be eye forms broken because of the fragility of single-strand regions at the fork. Examples of several partially replicated molecules are shown in Fig. 2. Figure 2a shows a T7 length molecule with an internal eye form of 13.7%. Internally replicated regions spanning 1 to 25% of unit T7 length were found. Figure 2b is a molecule with a large equal-armed fork. In addition to large forks spanning up to 38% of the molecule, small forks occurring at one end were found (Fig. 2c). A small number of molecules containing both a fork at one end and an internally duplicated region at the same end (Fig. 2d) were observed. Sixty-four partially replicated molecules were photographed and measured. Only molecules measuring 12.0 ± 0.5 ym (T7 unit length under our condition) were included in the analysis. Molecules were normalized to a scale of 100 units and aligned such that all partially replicated regions occurred in the same end (Fig. 3). We feel that it is reasonable to assume that these regions are all located at one end, the genetic left end of the molecule, for the following reasons. (i) Progeny DNA produced by infection with UV-irradiated T7+ phage containing six to seven phage lethal events per genome hybridized to restriction fragments from the left portion of the T7+ molecule but not to fragments from the right side (3). (ii) Wolfson et al. (22) and Dressler et al. (7) examined partial denaturation maps of partially replicated nonirradiated T7+ DNA and found that Y and eye
I=1 Z
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FRACTION NUMBER
FIG. 1. CsCl density gradient analysis ofpartially replicated T7 DNA. E. coli were grown in densitylabeled medium and infected with UV-irradiated T7 as described in the text. Intracellular DNA was extracted and banded in a CsCl density gradient. DNA displaced from the unreplicated, parental T7 DNA toward the heavy location of the gradient was isolated and rebanded in a second gradient. This figure shows the distribution of the DNA in the second gradient. The material in the fractions from the second gradient indicated by brackets was pooled for electron microscopy. Symbols: (L-F) 3H counts per minute x 10-3 (E. coli DNA); (- -0) 32p counts per minute x 10-3 (T7 DNA).
forms were located on the left side of the molecule. They mapped an origin of T7 DNA replication at a location 17% from the left end of the T7 genome. (iii) Four molecules containing both a fork and an eye form were observed, and these regions were both located in the same end of the molecule (ii) No molecules containing partially replicated regions at both ends were seen. It is evident from the distribution of eye forms over the left 20% of the molecule that no single origin of replication was utilized (Fig. 3). If replication is assumed to proceed bidirectionally at the same rate from the origin of replication of a molecule, the distribution of origins among our eye forms (including unequal forks and eyes in multiple structures) is given in Table 1. In addition, there are eight molecules with forks spanning 0 to 5% of the molecule and an additional six spanning 0 to 10% of the genome. Therefore, initiation can proceed from a point at or near the left end of a UV-irradiated T7 DNA molecule, as well as from several locations along the left 30% of the genome. The summed total of partially replicated re-
ELECTRON MICROSCOPY OF T7 DNA
VOL. 32, 1979
609
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gions observed in our molecules is given by the histogram in Fig. 4; 75% of the partially replicated areas lie to the left of 17%, whereas only 25% are to the right of that point. This curve closely resembles that obtained for marker rescue experiments, using T7+ irradiated with a similar dose (3) (Fig. 4). The similarity of the two curves implies that partial replication of the left end of a UV-irradiated T7+ genome can account for the distribution of marker rescue efficiency and supports our estimations of partial replication as determined by DNA hybridization. Effect of photoreactivation on marker rescue. The formation of pyrimidine dimers is an important type of UV radiation-induced damage to DNA (16, 17, 19). Several lines of evidence have established that pyrimidine dimers are blocks to DNA synthesis (9, 11, 15). Illumination of UV-irradiated bacteria with photoreactiving light results in the removal of thymine dimers, the resumption of blocked DNA synthesis, and the ultimate recovery of the UV-irradiated cells (15, 16, 18). Our interpretation of cross-reactivation experiments with bacteriophage T7 is that thymine dimers to the right of the origin of
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T7 DNA replication block further replication to the right, thereby inhibiting marker rescue in the right end of the T7 genome. The distribution of partially replicated regions described in this paper and previously (3), therefore, reflect the chance that thymine dimers interrupt the progress of the replication fork toward the right end of the T7 DNA molecule. Consequently, one should detect a large difference in the marker rescue patterns resulting from cross-reactivation experiments performed in dim light versus those performed in photoreactivating light. Not only should the absolute level of wild-type infective centers increase, but also the overall pattern should broaden toward the right end of the genetic map as thymine dimers are removed, thus allowing replication to proceed further to the right. To test the hypothesis outlined above, we coinfected E. coli with UV-irradiated T7+ phage and one of a series of T7 amber mutants. The infective centers were divided into two portions; one part was handled continuously in dim or no light, and one part was illuminated with General Electric F40 fluorescent lights. The results of the experiment are shown in Fig. 5. As expected,
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BURCK, SCRABA, AND MILLER
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FIG. 3. Line diagrams of partially replicated T7 molecules. Partially replicated molecules were measured and normalized to unit length. All of the branches of the molecules indicated in these drawings were double stranded, but we did not undertake a comprehensive analysis of very small single-stranded regions (a few hundred nucleotides), possible at forks, for example. TABLE 1. Distribution of eye form centers" No. of eye midpoints
Segment of T7 DNA (% length) 0-5 5-10 10-15
15-20 >20
6 9 10 9 7
The midpoints of the eye forms represented by the line drawings of Fig. 3 were measured. This table lists the number of times an eye form midpoint falls within a particular segment of T7 DNA among the molecules diagrammed in Fig. 3.
removing thymine dimers from the DNA by photoreactivation changed the overall pattern of cross-reactivation; regions toward the right end
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FIG. 4. Histogram of partially replicated regions from UV-irradiated T7 DNA. Lengths of T7+ DNA in 1% increments were scored for replication as represented in the line drawings of Fig. 3. The histogram shows the number of times a particular segment of DNA was replicated in the pool of molecules diagrammed in Fig. 3. The open circles represent marker rescue frequencies normalized to the maximum rescue and distributed along the genetic map of T7 as a function of a percent length of genome (3).
of the map were rescued with a greater relative efficiency. The absolute levels of marker rescue increased slightly for all portions of the genome after photoreactivation, but the dramatic change diated T7+ DNA. Partial replicas of bacteriooccurred in the pattern of marker rescue toward phage DNA originally were postulated on gethe right end of the map. The results of this netic grounds to account for the patterns of experiment were consistent with our hypothesis cross-reactivation and multiplicity reactivation that thymine dimers normally block replication observed with bacteriophage T4 (1, 6). Several to the right. As thymine dimers were removed, lines of evidence are consistent with the hypothreplication proceeded to the right and more esis. In mixed infection by UV-irradiated T4D+ marker rescue occurred to the right. phage and different members of a set of defined T4D mutants, the ability of a particular mutant DISCUSSION to rescue the damaged T4D+ genome was deIn this paper, we present direct visual evidence pendent on the map position of the marker (23). for the existence of partial replicas of UV-irra- Four distinct peaks were obtained in this exper-
VOL. 32, 1979
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611
and the ability of a particular segment of the genome to be efficiently rescued. Several features of bacteriophage T7 have facilitated an examination of the relationships among marker rescue, partial replication of UVirradiated DNA, and origins of DNA replication. An origin of replication was mapped for T7+ DNA by electron microscopic analysis of parIC tially replicated (non-irradiated) T7+ molecules (7). Restriction fragments of the T7 genome 0.3 1 C 13 1 5 8 were aligned with specific regions of the genetic 17 I 14 11 0.7 1lb 2 4 6 16 19 map (12). We showed that partial replicas of T7 DNA are produced in E. coli infected with UVMAP POSITION irradiated bacteriophage by hybridizing 32P-labeled progeny DNA from such an infection to ordered T7 restriction fragments. We showed further that the segments of the UV-irradiated l l | T7 DNA which replicated efficiently were those I I 20 60 40 80 0 10 which rescued efficiently during cross-reactivation experiments (3). Partial replicas of UV-irPERCENT LENGTH OF GENOME radiated DNA were thought to result from the FIG. 5i. Effect ofphotoreactivation on marker res- blockage of replication at UV-damaged regions. cue. E. coli 011 ' (supE) was grown to 3 x 108 bacteria Replication originally was thought to be initiper ml,in H-broth at 300C. Chloramphenicol was ated 17% from the left end of the irradiated T7 added act 100 pg/ml to the culture to inhibit superin- genome and to proceed bidirectionally at the fection eexclusion (2). At 1-min intervals, samples of same rate to the ends of the molecule or to the the cells were coinfected with UV-irradiated T7' to the est sites tof UV phage (r^eceiving 10.1 phage lethal events) and one of nearest sites of UV damage. On this basis, a series of T7 amber phage. The multiplicity of infec- markers around 17% on the genome should be tion of ecach type was close to five phage per bacterium rescued most efficiently. However, we found that and was monitored by plating the surviving bacteria. at higher radiation doses, markers to the left of (Survivi?ng bacteria were monitored in parallel cul- 17% were rescued with consistently greater effitures in,fected with UV-irradiated T7+ alone and ciency than were markers to the right of 17% (3). with T7 mutants alone.) At 10 min after infection, a Analysis of the progeny DNA produced by UVsample cgf the infected cells was diluted into T7 anti- irradiated T7+ parental phage indicated that serum, ijncubated for 5 min at 37°C, diluted further, irradite of parenta th at and platted for infective centers on E. coli B23 (sup). segments of the genome to the left of 17% also Infective -center production is not significantly influ- were replicated with greater efficiency than were enced byy incubation in chloramphenicol for up to 30 those to the right. However, the location and min. Two sequential experiments using the same UV- size of the restriction fragments used in the irradiat edphage were performed. One was performed analysis were such that a fine-structure mapping in the d 'ark, and plates were incubated in the dark. of the partial replicas was not possible. The The othter was performed with the room (fluorescent) electron microscopic data presented here show lights orz, and plates were incubated for 15 h under that more DNA to the left of 17% is replicated fluoresc ent lamps. All of the amber phage represented than to the right (Fig. 4), as expected from on each curve were examined in a single experiment. marker rescue data. la, Ib, a nd Ic stand for ambers lamI93, lam323, and One limitation of the previous studies on the lam342cx, respectively. Symbols: (O---O-) no photoOelaino h rvossuiso h extent of partial replication (3) was the inability reactiva,tion; (e- ) with photoreactivation. to discriminate between DNA synthesis due to iment,;a number consistent with the number of replication and that due to repair. 32P-labeled origins thought to operate during T4 replication deoxynucleoside triphosphates would have been (5, 10). Rayssiguier and Vigier (14) analyzed the incorporated into UV-irradiated DNA by both recombinant clone size distribution of progeny processes. The incorporation due to repair synphage Iproduced by multiplicity reactivation of thesis certainly would have been more random UV-irraLdiated genetically marked parental and, consequently, would have contributed to phage. Their analysis was consistent with the radioactive DNA annealing along the whole of idea th at partial replicas of the damaged ge- the T7 genome. In fact, estimates of the extent nomes:reassociate by recombining primarily at of replication indicated that portions of the right their esatremities. These experiments suggested end of the UV-irradiated molecules might be a correlLation between origins of DNA replication labeled to a greater extent than they were resw
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cued during cross-reactivation studies. This could have been due to the presence of some repair synthesis. The direct visualization of partially replicated regions in an electron microscope allows one to score for replicated regions, as opposed to regions of repair synthesis. The excellent correlation between the histogram of replicated regions and the distribution of marker rescue frequencies supports very well our previous conclusions (Fig. 4 and reference 3). One implication of our previous results is that the replication of UV-irradiated T7+ DNA is initiated at an origin or origins to the left of 17%. At the same time, additional interpretations also are possible. Since the primary mechanism of UV radiation damage to DNA is the formation of pyrimidine, and especially thymidine, dimers (16, 17, 19), regions of the DNA rich in adeninethymine base pairs would be expected to sustain more lethal damage than would regions where guanine-cytosine base pairs predominate. Examination of the partial denaturation map of T7 (8) shows that adenine-thymine-rich regions are not uniformly distributed along the T7 molecules. The region from - 10 to 30% containing the presumed DNA replication origin site at 17% is very rich in adenine-thymine base pairs, whereas the region from around 5 to 10% is practically devoid of sites susceptible to partial denaturation. With this consideration, the marker rescue and hybridization data could be consistent with bidirectional replication from an origin located around 17% from the left end until a site of UV damage is reached. Such sites would be more probable to the right than to the left of the origin. However, analysis of partially replicated T7+ molecules with an electron microscope supports the conclusion that UV-irradiated T7 molecules are able to initiate replication at a number of sites within the left end of the molecule (Fig. 3), most of which are located to the left of 17% (Table 1). The timing of the experiment allowed replication to proceed as far as possible, so one cannot say exactly where along an eye replication started since replication stops at a UV lesion on either side of the initiation site; that is, replication did not necessarily initiate at the center of the eye. Unirradiated molecules may not behave in the same way; i.e., it is entirely possible that normal molecules initiate primarily at the 17% origin, but that UVirradiated molecules utilize secondary sites along the left end when damage is sustained in the 17% region. An examination of the distribution of replicated regions reveals that UV-irradiated molecules are replicated primarily to the left of 17% (Fig. 4), since 75% of the partially replicated
regions observed were to the left of 17% whereas only 25% were to the right. It is not surprising that most of the partially replicated regions lie to the left of 17% since, on a probability basis, UV damage will occur to a greater extent in the adenine-thymine-rich region around and to the right of 17%. The photoreactivation data presented here (Fig. 5) support this view. As thymine dimers are removed from UV-irradiated T7 DNA, the pattern of markers rescued broadens toward the right end of the genome; when photoreactivation occurs, markers to the right of 17% are rescued with a higher efficiency. This is consistent with our hypothesis that thymine dimers normally block replication to the right, thus inhibiting marker rescue in this direction. In conclusion, the electron micrographic analysis confirms and extends our previous results (3). There is an excellent correlation between the areas of the genome which replicate efficiently and those which are rescued efficiently during cross-reactivation. Furthermore, origins other than that at 17% are used to replicate UVirradiated DNA. On the other hand, the marker rescue patterns obtained during cross-reactivation experiments probably best represent the final extent of partial replication rather than the exact location of origins of DNA replication. ACKNOWLEDGMENTS This research was supported by grants from the National Research Council and the Medical Research Council of Canada. K.B.B. was supported by fellowships from the Killam Foundation and the Medical Research Council of Canada. We thank W. F. Studier for the bacteriophage used in this study, A. H. Doermann and C. C. Richardson for helpful suggestions, and Roger Bradley, Deborah Taylor, and Helen Smith for technical assistance.
LITERATURE CITED 1. Barricelli, N. A., and A. H. Doermann. 1961. An analytical approach to the problems of phage recombination and reproduction. III. Cross-reactivation. Virology 13:460-476. 2. Benbasat, J., K. B. Burck, and R. C. Miller, Jr. 1978. Superinfection exclusion and lack of conservative transfer of bacteriophage T7 DNA. Virology 87:164-171. 3. Burck, K. B., and R. C. Miller, Jr. 1978. Marker rescue and partial replication of bacteriophage T7 DNA. Proc. Natl. Acad. Sci. U.S.A. 75:6144-6148. 4. Davis, R. W., M. Simon, and N. Davidson. 1971. Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids. Methods Enzymol. 21:413-428. 5. Delius, H., C. Howe, and A. W. Kozinski. 1971. Structure of the replicating DNA from bacteriophage T4. Proc. Natl. Acad. Sci. U.S.A. 68:3049-3053. 6. Doermann, A. H., M. Chase, and F. W. Stahl. 1955. Genetic recombination and replication in bacteriophage. J. Cell. Comp. Physiol. 45(Suppl.):51-74. 7. Dressler, D., J. Wolfson, and M. Magazin. 1972. Initiation and reinitiation of DNA synthesis during replication of bacteriophage T7. Proc. Natl. Acad. Sci. U.S.A. 69:998-1002.
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8. Gomez, B., and D. Lang. 1972. Denaturation map of bacteriophage T7 DNA and direction of DNA transcription. J. Mol. Biol. 70:239-251. 9. Hourcade, D., and D. Dressler. 1978. Site-specific initiation of a DNA fragment. Proc. Natl. Acad. Sci. U.S.A. 75:1652-1656. 10. Howe, C. C., P. J. Buckley, K. Carlson, and A. W. Kozinski. 1973. Multiple and specific initiation of T4 DNA replication. J. Virol. 12:130-148. 11. Masamune, Y. 1976. Effect of ultraviolet irradiation of bacteriophage Fl DNA on its conversion to replicative form by extracts of Escherichia coli. Mol. Gen. Genet. 149:335-345. 12. McDonnel, M. W., M. N. Simon, and F. W. Studier. 1977. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mol. Biol. 110:119-146. 13. Miller, R. C. Jr., M. Lee, D. G. Scraba, and V. Paetkau. 1976. The role of bacteriophage T7 exonuclease (gene 6) in genetic recombination and production of concatemers. J. Mol. Biol. 101:223-234. 14. Rayssiguier, C., and P. R. R. Vigier. 1977. Genetic evidence for the existence of partial replicas of T4 genomes inactivated by irradiation under ultraviolet light. Virology 78:442-452.
15. Rupp, W., and P. Howard-Flanders. 1968. Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J. Mol. Biol. 31:291-304. 16. Setlow, J. K. 1966. The molecular basis of biological effects of ultraviolet radiation and photoreactivation. Curr. Top. Radiat. Res. 2:195-248. 17. Setlow, R. B. 1964. Physical changes and mutagenesis. J. Cell. Comp. Physiol. 64(Suppl. 1):51-68. 18. Setlow, R. B. 1968. The photochemistry, photobiology, and repair of polynucleotides. Prog. Nucleic Acid Res. Mol. Biol. 8:257-295. 19. Setlow, R. B., and W. L. Carrier. 1966. Pyrimidine dimers in ultraviolet-irradiated DNA. J. Mol. Biol. 17: 237-254.
20. Studier, F. W. 1969. The genetics and physiology of bacteriophage T7. Virology 39:562-574. 21. Studier, F. W. 1973. Genetic analysis of non-essential bacteriophage T7 genes. J. Mol. Biol. 79:227-236. 22. Wolfson, J. D., D. Dressler, and M. Magazin. 1972. Bacteriophage T7 DNA replication: a linear replicating intermediate. Proc. Natl. Acad. Sci. U.S.A. 69:499-504. 23. Womack, F. C. 1965. Cross-reactivation differences in bacteriophage T4 D. Virology 26:758-761.
JOURNAL OF VIROLOGY, Nov. 1979, p. 606-613
0022-538X/79/11-0606/08$02.00/0
Electron Microscopic Analysis of Partially Replicated Bacteriophage T7 DNA KATHY BAUMAN BURCK,1 DOUGLAS G. SCRABA,2 AND ROBERT C. MILLER, JR.`* Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1 W5,1 and Department of Biochemistry, University ofAlberta, Edmonton, Alberta, Canada2 Received for publication 30 May 1979
Partially replicated bacteriophage T7 DNA was isolated from Escherichia coli infected with UV-irradiated T7 bacteriophage and was analyzed by electron microscopy. The analysis determined the distribution of eye forms and forks in the partially replicated molecules. Eye forms and forks in unit length molecules were aligned with respect to the left end of the T7 genome, and segments were scored for replication in each molecule. The resulting histogram showed that only the left 25 to 30% of the molecules was replicated. Several different origins of DNA replication were used to initiate replication in the UV-irradiated molecules. The results are in excellent agreement with those of hybridization experiments in which 32P-labeled progeny DNA from UV-irradiated phage was annealed with ordered restriction fragments of T7 DNA (K. B. Burck and R. C. Miller, Jr., Proc. Natl. Acad. Sci. U.S.A. 75:6144-6148, 1978). Both analyses support partial-replica hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460-476, 1961; Doermann et al., J. Cell. Comp. Physiol. 45[Suppl.]:51-74, 1955) as an explanation for the distribution of marker rescue frequencies during cross-reactivation; i.e., replication proceeds in a bidirectional manner from an origin to a site of UV damage, and those regions of the genome which replicate most efficiently are rescued most efficiently by a coinfecting phage. In addition, photoreactivation studies support the hypothesis that thymine dimers are the major UV damage blocking cross-reactivation in the right end of the T7 genome.
Cross-reactivation refers to a process whereby genetic markers are rescued from a UV-irradiated, wild-type phage by a coinfecting mutant phage. The probability of a specific marker being rescued during T4 or T7 phage infection depends on the map position of the marker (3, 23). During T7 infection, only markers toward the left end of the molecules are rescued efficiently. Evidence has been presented which indicates that those markers which are rescued efficiently are markers which replicate efficiently (3); i.e., 32P_ labeled progeny DNA synthesized after infection by a UV-irradiated T7+ phage hybridizes predominantly with restriction fragments of T7+ DNA known to carry markers rescued efficiently during cross-reactivation. The effect is dose dependent: the higher the dose of UV irradiation, the fewer the markers which are rescued efficiently and the smaller the area of the genome which replicates efficiently. One idea which correlates the efficiency of replication with the efficiency of marker rescue is the partial-replica hypothesis (1, 6). This theory postulates that replication of a UV-irradiated genome starts from a specific origin(s) and proceeds in a bidirectional manner to UV lesions
which block further replication. Subsequently, those regions of the genome which have replicated most efficiently are rescued most efficiently by a coinfecting phage. The hybridization data mentioned above are in agreement with this hypothesis, the data clearly indicating the presence of partial replicas of UV-irradiated genomes after infection. This report presents the results of an electron microscopic analysis of partially replicated, UVirradiated T7+ DNA. It describes the distribution of replicated regions, growing forks, and bubbles in replicating, UV-irradiated T7+ DNA isolated from infected cells. In addition, a photoreactivation experiment is reported here which supports the hypothesis that thymine dimers normally block cross-reactivation in the right end of the T7 map. All of the results agree with and extend our previous conclusions and strongly support the partial-replica hypothesis. MATERIALS AND METHODS
606
Bacterial and phage strains. Escherichia coli B23 (sup°) was used as the nonpermissive host for cross-reactivation experiments. After adaptation for growth in density medium (see below), B23 was used as the host for the isolation of partially replicated T7+
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DNA for electron microscopy. E. coli 011' (supE) was used as the permissive host during cross-reactivation experiments. All phage strains originally were provided by F. W. Studier. Our T7+ (wild-type) strain was determined to be free of deletions and like the original Studier strain by restriction endonuclease (HpaI and MboI) analysis (12). Namx is the general designation for amber mutants, where N is the gene number, and x is the specific mutant. The mutants used in this study were: lam193, lam323, lam342a, 2am64, 3am29, 4am208, 5am28, 6am233, 8amll, 11am37, 14am140, 16am194, 17am290, and l9amlO (20). Chemicals and isotopes. Thymidine was purchased from Worthington Biochemicals Corp. Uracil, 5-fluorodeoxyuridine, and cytochrome c (type V) were from Sigma Chemical Co. The density isotopes 2H20, '5NH4Cl, and deuterated algal whole hydrolysate were from Merck Sharp & Dohme. [methyl-3H]thymidine and 32P as H332PO4 were purchased from New England Nuclear Corp. Formamide was from Matheson, Coleman and Bell and was deionized before use by treatment with Bio-Rad AG 501-X8 mixed-bed resin. Media and buffers. Density medium contained the following (per liter of 2H20): 7.0 g of Na2HPO4, 3.0 g of KH2PO4, 1.0 g of '5NH4Cl, 0.5 g of NaCl, and 0.03 ml of 0.1 M FeCli. After autoclaving, the following were added: 1.0 ml of 1 M MgSO4, 0.1 ml of 1 M CaCl2, 12.5 ml of 20% glucose in 2H20, and 1.12 ml of deuterated algal whole hydrolysate (22). Tris-NaCl-EDTA buffer (TNE) contained 0.01 M Tris-hydrochloride, 0.15 M NaCl, and 0.015 M EDTA (pH 7.4). Tris-EDTA buffer contained 0.5 M Trishydrochloride and 0.05 M EDTA (pH 7.0). Lysis buffer contained 0.1 M NaCl, 0.02 M EDTA, 0.01 M KCN, 0.01 M iodoacetate, and 0.1 M Tris (pH 7.4) (22). T7 Tris salt was 1 M NaCl and 0.05 M Tris-hydrochloride
to be 12.0 + 0.5 [Lm in these experiments). Isolation of partially replicated molecules after infection of E. coli B23 by UV-irradiated T7+ phage. E. coli B23 was adapted for growth in the density medium by serial passage through 20, 40, 60, 75, 90, 95, and 100% substituted medium (22). The adapted cells had a generation time of 75 to 80 min at 37°C. Non-irradiated T7+ phage followed a normal single-step growth curve at 30°C in the density-labeled cells, giving a burst of 120 phage per cell by 45 min after infection. A 50-ml culture of density-labeled E. coli B23 was grown to approximately 3 x 108 cells per ml at 37°C in heavy medium containing 5 jig of thymidine per ml, 5, g of 5-fluorodeoxyuridine per ml, 25 ,ug of uracil per ml, and 500 ,uCi of [methyl-'H]thymidine and then shifted to 30°C for 15 min. This procedure allowed labeling of the bacterial cells for HH reference (both strands labeled) in CsCl gradients. The cells then were infected at a multiplicity of infection of 2 with UV-irradiated, 32P-labeled LL (no density label in either strand) T7+ containing 7.3 lethal events per genome. The 32p label was at a specific activity of 2 mCi/mg, which leads to the incorporation of a 32p atom in two-fifths of the phage particles. The multiplicity of infection was determined by monitoring surviving bacteria. Since no attempt was made in this experiment to overcome superinfection exclusion (2), a calculated multiplicity of infection of 2 does not necessarily mean that any cells received injected DNA from more than one phage. Infective centers and background phage also were monitored. UV-irradiated T7 phage are unable to conduct a productive infection: infective centers are typically only 0.1 to 0.2% of the control bacteria. Non-irradiated T7+ produce infective centers on 90% of the control bacteria. At 25 nmin postinfection, the culture was chilled by pipetting it into 2 volumes of ice-cold TNE plus 1 volume of lysis buffer. The infected cells were sedimented and resuspended at a concentration of 1.5 x 109 bacteria per ml in lysis buffer. Cells then were lysed with lysozyme (400,ug/ml, 0°C, 45 min) followed by detergent (0.1% sodium lauryl sarcosinate, 65°C, 20 min). The lysate was deproteinized by treatment with self-digested pronase (1 mg/ml, 37°C, 12 h). Samples of the lysate were mixed 1:4 with saturated CsCl (in distilled water) and centrifuged in an SB 283 rotor in a B60 International centrifuge at 30,000 rpm for 72 h at 12°C. Approximately 70 fractions were collected from the bottom of the tube and assayed for ;'H (HH E. coli DNA) and 32p (T7 DNA). Fractions banding on the heavy side of the T7 (P2P) peak were pooled and centrifuged in a Beckman type 65 rotor at 32,000 rpm for 72 h at 12°C. Specific fractions from the second gradient were
(pH 7.4).
UV irradiation of phage. T7+ phage were diluted
to 10" bacteriophage per ml in T7 Tris salt and placed in a plastic petri dish on ice. Irradiation was for 20 s at a distance of 30 cm from a General Electric G14T8 15W germicidal lamp. At this dose, T7 phage received approximately 7.3 lethal hits per particle. Lethal events were quantitated by plotting a survival curve
of the irradiated phage. Cross-reactivation and photoreactivation. Cross-reactivation experiments were performed as previously described (3). When photoreactivation was desired, cross-reactivation was performed under fluorescent-light illumination, and infective centers were incubated beneath General Electric F40 fluorescent lights for 15 h at room temperature. Electron microscopy. Selected fractions from CsCl gradients were dialyzed overnight (4°C) against 0.1 M Tris-10 mM EDTA (pH 7.0) before being mounted for electron microscopic examination by the 40%-10% formamide spreading procedure of Davis et al. (4). Partially replicated molecules were photographed at a magnification of x44,000 with a Phillips EM300 electron microscope operated at 60 kV and with a 30-nm-objective aperture. Molecular lengths were determined with a map measuring device from tracings of prints enlarged three times. All measurements were normalized to unit length T7 DNA (found
pooled for electron microscopy. Before centrifugation, the polyallomer tubes were treated for 1 h with 10% bovine serum albumin. Other methods. Preparation of '32P-labeled T7 bacteriophage and determination of label uptake into acid-insoluble material have been described previously
(13).
RESULTS Isolation of partially replicated T7+ DNA. Partially replicated T7 DNA molecules were
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isolated from density-labeled E. coli infected with UV-irradiated phage as described above. The DNA was sedimented to equilibrium in CsCl density gradients, and the distribution of radioactivity was determined. As expected, T7+ irradiated to 7.3 phage lethal events did not undergo even one round of replication as no parental DNA banded at the hybrid location. Material banding on the heavy side of the T7 DNA peak was pooled and resedimented in a second CsCl gradient (Fig. 1). The fractions of this gradient indicated by brackets were pooled and analyzed by electron microscopy. This figure shows that the T7 DNA was well separated from contaminating E. coli DNA. Electron microscopy of partially replicated T7+ DNA. Partially replicated, UV-irradiated T7+ DNA isolated from a CsCl gradient was examined by electron microscopy. Four categories of partially replicated molecules of T7 size DNA were distinguished: (i) eye forms, (ii) molecules containing forks with two equal arms, (iii) mrolecules containing both eye forms and forks, and (iv) molecules containing forks with arms of unequal length, where two of the branches together were of T7 length; class iv molecules were assumed to be eye forms broken because of the fragility of single-strand regions at the fork. Examples of several partially replicated molecules are shown in Fig. 2. Figure 2a shows a T7 length molecule with an internal eye form of 13.7%. Internally replicated regions spanning 1 to 25% of unit T7 length were found. Figure 2b is a molecule with a large equal-armed fork. In addition to large forks spanning up to 38% of the molecule, small forks occurring at one end were found (Fig. 2c). A small number of molecules containing both a fork at one end and an internally duplicated region at the same end (Fig. 2d) were observed. Sixty-four partially replicated molecules were photographed and measured. Only molecules measuring 12.0 ± 0.5 ym (T7 unit length under our condition) were included in the analysis. Molecules were normalized to a scale of 100 units and aligned such that all partially replicated regions occurred in the same end (Fig. 3). We feel that it is reasonable to assume that these regions are all located at one end, the genetic left end of the molecule, for the following reasons. (i) Progeny DNA produced by infection with UV-irradiated T7+ phage containing six to seven phage lethal events per genome hybridized to restriction fragments from the left portion of the T7+ molecule but not to fragments from the right side (3). (ii) Wolfson et al. (22) and Dressler et al. (7) examined partial denaturation maps of partially replicated nonirradiated T7+ DNA and found that Y and eye
I=1 Z
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FRACTION NUMBER
FIG. 1. CsCl density gradient analysis ofpartially replicated T7 DNA. E. coli were grown in densitylabeled medium and infected with UV-irradiated T7 as described in the text. Intracellular DNA was extracted and banded in a CsCl density gradient. DNA displaced from the unreplicated, parental T7 DNA toward the heavy location of the gradient was isolated and rebanded in a second gradient. This figure shows the distribution of the DNA in the second gradient. The material in the fractions from the second gradient indicated by brackets was pooled for electron microscopy. Symbols: (L-F) 3H counts per minute x 10-3 (E. coli DNA); (- -0) 32p counts per minute x 10-3 (T7 DNA).
forms were located on the left side of the molecule. They mapped an origin of T7 DNA replication at a location 17% from the left end of the T7 genome. (iii) Four molecules containing both a fork and an eye form were observed, and these regions were both located in the same end of the molecule (ii) No molecules containing partially replicated regions at both ends were seen. It is evident from the distribution of eye forms over the left 20% of the molecule that no single origin of replication was utilized (Fig. 3). If replication is assumed to proceed bidirectionally at the same rate from the origin of replication of a molecule, the distribution of origins among our eye forms (including unequal forks and eyes in multiple structures) is given in Table 1. In addition, there are eight molecules with forks spanning 0 to 5% of the molecule and an additional six spanning 0 to 10% of the genome. Therefore, initiation can proceed from a point at or near the left end of a UV-irradiated T7 DNA molecule, as well as from several locations along the left 30% of the genome. The summed total of partially replicated re-
ELECTRON MICROSCOPY OF T7 DNA
VOL. 32, 1979
609
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gions observed in our molecules is given by the histogram in Fig. 4; 75% of the partially replicated areas lie to the left of 17%, whereas only 25% are to the right of that point. This curve closely resembles that obtained for marker rescue experiments, using T7+ irradiated with a similar dose (3) (Fig. 4). The similarity of the two curves implies that partial replication of the left end of a UV-irradiated T7+ genome can account for the distribution of marker rescue efficiency and supports our estimations of partial replication as determined by DNA hybridization. Effect of photoreactivation on marker rescue. The formation of pyrimidine dimers is an important type of UV radiation-induced damage to DNA (16, 17, 19). Several lines of evidence have established that pyrimidine dimers are blocks to DNA synthesis (9, 11, 15). Illumination of UV-irradiated bacteria with photoreactiving light results in the removal of thymine dimers, the resumption of blocked DNA synthesis, and the ultimate recovery of the UV-irradiated cells (15, 16, 18). Our interpretation of cross-reactivation experiments with bacteriophage T7 is that thymine dimers to the right of the origin of
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T7 DNA replication block further replication to the right, thereby inhibiting marker rescue in the right end of the T7 genome. The distribution of partially replicated regions described in this paper and previously (3), therefore, reflect the chance that thymine dimers interrupt the progress of the replication fork toward the right end of the T7 DNA molecule. Consequently, one should detect a large difference in the marker rescue patterns resulting from cross-reactivation experiments performed in dim light versus those performed in photoreactivating light. Not only should the absolute level of wild-type infective centers increase, but also the overall pattern should broaden toward the right end of the genetic map as thymine dimers are removed, thus allowing replication to proceed further to the right. To test the hypothesis outlined above, we coinfected E. coli with UV-irradiated T7+ phage and one of a series of T7 amber mutants. The infective centers were divided into two portions; one part was handled continuously in dim or no light, and one part was illuminated with General Electric F40 fluorescent lights. The results of the experiment are shown in Fig. 5. As expected,
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BURCK, SCRABA, AND MILLER
-0 n
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FIG. 3. Line diagrams of partially replicated T7 molecules. Partially replicated molecules were measured and normalized to unit length. All of the branches of the molecules indicated in these drawings were double stranded, but we did not undertake a comprehensive analysis of very small single-stranded regions (a few hundred nucleotides), possible at forks, for example. TABLE 1. Distribution of eye form centers" No. of eye midpoints
Segment of T7 DNA (% length) 0-5 5-10 10-15
15-20 >20
6 9 10 9 7
The midpoints of the eye forms represented by the line drawings of Fig. 3 were measured. This table lists the number of times an eye form midpoint falls within a particular segment of T7 DNA among the molecules diagrammed in Fig. 3.
removing thymine dimers from the DNA by photoreactivation changed the overall pattern of cross-reactivation; regions toward the right end
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FIG. 4. Histogram of partially replicated regions from UV-irradiated T7 DNA. Lengths of T7+ DNA in 1% increments were scored for replication as represented in the line drawings of Fig. 3. The histogram shows the number of times a particular segment of DNA was replicated in the pool of molecules diagrammed in Fig. 3. The open circles represent marker rescue frequencies normalized to the maximum rescue and distributed along the genetic map of T7 as a function of a percent length of genome (3).
of the map were rescued with a greater relative efficiency. The absolute levels of marker rescue increased slightly for all portions of the genome after photoreactivation, but the dramatic change diated T7+ DNA. Partial replicas of bacteriooccurred in the pattern of marker rescue toward phage DNA originally were postulated on gethe right end of the map. The results of this netic grounds to account for the patterns of experiment were consistent with our hypothesis cross-reactivation and multiplicity reactivation that thymine dimers normally block replication observed with bacteriophage T4 (1, 6). Several to the right. As thymine dimers were removed, lines of evidence are consistent with the hypothreplication proceeded to the right and more esis. In mixed infection by UV-irradiated T4D+ marker rescue occurred to the right. phage and different members of a set of defined T4D mutants, the ability of a particular mutant DISCUSSION to rescue the damaged T4D+ genome was deIn this paper, we present direct visual evidence pendent on the map position of the marker (23). for the existence of partial replicas of UV-irra- Four distinct peaks were obtained in this exper-
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and the ability of a particular segment of the genome to be efficiently rescued. Several features of bacteriophage T7 have facilitated an examination of the relationships among marker rescue, partial replication of UVirradiated DNA, and origins of DNA replication. An origin of replication was mapped for T7+ DNA by electron microscopic analysis of parIC tially replicated (non-irradiated) T7+ molecules (7). Restriction fragments of the T7 genome 0.3 1 C 13 1 5 8 were aligned with specific regions of the genetic 17 I 14 11 0.7 1lb 2 4 6 16 19 map (12). We showed that partial replicas of T7 DNA are produced in E. coli infected with UVMAP POSITION irradiated bacteriophage by hybridizing 32P-labeled progeny DNA from such an infection to ordered T7 restriction fragments. We showed further that the segments of the UV-irradiated l l | T7 DNA which replicated efficiently were those I I 20 60 40 80 0 10 which rescued efficiently during cross-reactivation experiments (3). Partial replicas of UV-irPERCENT LENGTH OF GENOME radiated DNA were thought to result from the FIG. 5i. Effect ofphotoreactivation on marker res- blockage of replication at UV-damaged regions. cue. E. coli 011 ' (supE) was grown to 3 x 108 bacteria Replication originally was thought to be initiper ml,in H-broth at 300C. Chloramphenicol was ated 17% from the left end of the irradiated T7 added act 100 pg/ml to the culture to inhibit superin- genome and to proceed bidirectionally at the fection eexclusion (2). At 1-min intervals, samples of same rate to the ends of the molecule or to the the cells were coinfected with UV-irradiated T7' to the est sites tof UV phage (r^eceiving 10.1 phage lethal events) and one of nearest sites of UV damage. On this basis, a series of T7 amber phage. The multiplicity of infec- markers around 17% on the genome should be tion of ecach type was close to five phage per bacterium rescued most efficiently. However, we found that and was monitored by plating the surviving bacteria. at higher radiation doses, markers to the left of (Survivi?ng bacteria were monitored in parallel cul- 17% were rescued with consistently greater effitures in,fected with UV-irradiated T7+ alone and ciency than were markers to the right of 17% (3). with T7 mutants alone.) At 10 min after infection, a Analysis of the progeny DNA produced by UVsample cgf the infected cells was diluted into T7 anti- irradiated T7+ parental phage indicated that serum, ijncubated for 5 min at 37°C, diluted further, irradite of parenta th at and platted for infective centers on E. coli B23 (sup). segments of the genome to the left of 17% also Infective -center production is not significantly influ- were replicated with greater efficiency than were enced byy incubation in chloramphenicol for up to 30 those to the right. However, the location and min. Two sequential experiments using the same UV- size of the restriction fragments used in the irradiat edphage were performed. One was performed analysis were such that a fine-structure mapping in the d 'ark, and plates were incubated in the dark. of the partial replicas was not possible. The The othter was performed with the room (fluorescent) electron microscopic data presented here show lights orz, and plates were incubated for 15 h under that more DNA to the left of 17% is replicated fluoresc ent lamps. All of the amber phage represented than to the right (Fig. 4), as expected from on each curve were examined in a single experiment. marker rescue data. la, Ib, a nd Ic stand for ambers lamI93, lam323, and One limitation of the previous studies on the lam342cx, respectively. Symbols: (O---O-) no photoOelaino h rvossuiso h extent of partial replication (3) was the inability reactiva,tion; (e- ) with photoreactivation. to discriminate between DNA synthesis due to iment,;a number consistent with the number of replication and that due to repair. 32P-labeled origins thought to operate during T4 replication deoxynucleoside triphosphates would have been (5, 10). Rayssiguier and Vigier (14) analyzed the incorporated into UV-irradiated DNA by both recombinant clone size distribution of progeny processes. The incorporation due to repair synphage Iproduced by multiplicity reactivation of thesis certainly would have been more random UV-irraLdiated genetically marked parental and, consequently, would have contributed to phage. Their analysis was consistent with the radioactive DNA annealing along the whole of idea th at partial replicas of the damaged ge- the T7 genome. In fact, estimates of the extent nomes:reassociate by recombining primarily at of replication indicated that portions of the right their esatremities. These experiments suggested end of the UV-irradiated molecules might be a correlLation between origins of DNA replication labeled to a greater extent than they were resw
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BURCK, SCRABA, AND MILLER
cued during cross-reactivation studies. This could have been due to the presence of some repair synthesis. The direct visualization of partially replicated regions in an electron microscope allows one to score for replicated regions, as opposed to regions of repair synthesis. The excellent correlation between the histogram of replicated regions and the distribution of marker rescue frequencies supports very well our previous conclusions (Fig. 4 and reference 3). One implication of our previous results is that the replication of UV-irradiated T7+ DNA is initiated at an origin or origins to the left of 17%. At the same time, additional interpretations also are possible. Since the primary mechanism of UV radiation damage to DNA is the formation of pyrimidine, and especially thymidine, dimers (16, 17, 19), regions of the DNA rich in adeninethymine base pairs would be expected to sustain more lethal damage than would regions where guanine-cytosine base pairs predominate. Examination of the partial denaturation map of T7 (8) shows that adenine-thymine-rich regions are not uniformly distributed along the T7 molecules. The region from - 10 to 30% containing the presumed DNA replication origin site at 17% is very rich in adenine-thymine base pairs, whereas the region from around 5 to 10% is practically devoid of sites susceptible to partial denaturation. With this consideration, the marker rescue and hybridization data could be consistent with bidirectional replication from an origin located around 17% from the left end until a site of UV damage is reached. Such sites would be more probable to the right than to the left of the origin. However, analysis of partially replicated T7+ molecules with an electron microscope supports the conclusion that UV-irradiated T7 molecules are able to initiate replication at a number of sites within the left end of the molecule (Fig. 3), most of which are located to the left of 17% (Table 1). The timing of the experiment allowed replication to proceed as far as possible, so one cannot say exactly where along an eye replication started since replication stops at a UV lesion on either side of the initiation site; that is, replication did not necessarily initiate at the center of the eye. Unirradiated molecules may not behave in the same way; i.e., it is entirely possible that normal molecules initiate primarily at the 17% origin, but that UVirradiated molecules utilize secondary sites along the left end when damage is sustained in the 17% region. An examination of the distribution of replicated regions reveals that UV-irradiated molecules are replicated primarily to the left of 17% (Fig. 4), since 75% of the partially replicated
regions observed were to the left of 17% whereas only 25% were to the right. It is not surprising that most of the partially replicated regions lie to the left of 17% since, on a probability basis, UV damage will occur to a greater extent in the adenine-thymine-rich region around and to the right of 17%. The photoreactivation data presented here (Fig. 5) support this view. As thymine dimers are removed from UV-irradiated T7 DNA, the pattern of markers rescued broadens toward the right end of the genome; when photoreactivation occurs, markers to the right of 17% are rescued with a higher efficiency. This is consistent with our hypothesis that thymine dimers normally block replication to the right, thus inhibiting marker rescue in this direction. In conclusion, the electron micrographic analysis confirms and extends our previous results (3). There is an excellent correlation between the areas of the genome which replicate efficiently and those which are rescued efficiently during cross-reactivation. Furthermore, origins other than that at 17% are used to replicate UVirradiated DNA. On the other hand, the marker rescue patterns obtained during cross-reactivation experiments probably best represent the final extent of partial replication rather than the exact location of origins of DNA replication. ACKNOWLEDGMENTS This research was supported by grants from the National Research Council and the Medical Research Council of Canada. K.B.B. was supported by fellowships from the Killam Foundation and the Medical Research Council of Canada. We thank W. F. Studier for the bacteriophage used in this study, A. H. Doermann and C. C. Richardson for helpful suggestions, and Roger Bradley, Deborah Taylor, and Helen Smith for technical assistance.
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