52
Biocidmica et Biophysica Acta, ! 130(1992) 52-62 i.-
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Fig. 3. Densitometric scan of an autoradiogram which represents ~zP-labelled T7 D N A hybridized to T 7 / / p a l restriction fragments separated by electrophoresis on a !.2% agarose gel. Bacteriophage T7 containing "~'P-labellcd D N A was extensively dialyzed against 10 mM NaCI, 50 mM Tris-HCI (pH 8.0), 10 mM MgC!-, and the phage
DNA was purified and hybridized to T7 Hpal restriction fragments as described in Materials and Methods for the analysis of noninjeered DNA. The photograph of the autoradiogram was intentionally overexposed for publication.
INTENSITY (%)
Fig. 4. Quantitative analysis of hybridization between llpal T7 restriction fragments and T7 DNA. The relative intensity (~,:) of each band was compared to the expected relative intensity calculated from the molecular length of the corresponding ttpai u'estriction fragment [34]. Experimental intensities were obtained by averaging the results of four separate hybridization experiments. Average errors are indicated by the vertical lines while the broken diagonal line rcprescnt~ perfect confi~rmity of experimental and theoretical values.
indicating that extraction procedures and hybridization conditions permitted equivalent quantitative recovery of all parts of the genome. The intensity of band A was somewhat underestimated by our method. We believe this to be due to the fact that restriction fragment A, by far the largest of the Hpal products, did not elute from the agarose gel as fast as the other smaller fragments. By prolonging the transfer period (already about 15 h) we would have run the risk of losing the smallest fragments by elution out of and beyond the nitrocellulose sheet. Band A is thus very sensitive to transfer conditions and its intensity was considered significant only for hybridizations relating to the same initial gel. Calculation of the intensity of band E was also difficult because it is seen only as a shoulder of the F + G band in densitometric scans. In our analysis, we have combined the intensities of certain bands: L, M and N, C and D, and F + G and H (see Fig. 2). The first three correspond to three adjacent Hpal restriction fragments from the right end (last part to be injected) of the phage genome, while the F + G and H bands correspond to adjacent fragments from the left (first to be injected) end. The C and D bands correspond to adjacent restriction fragments situated just to the right of G on the Hpal restriction map of T7. By combining these bands, we have decreased the average error involved in the computer-determined attribution of peak-widths: M and N, C and D, as well as F + G and H are neighboring bands in the electrophoretic patterns and hence, contiguous peaks in the densitometric profiles. Unfortunately, band 1 + J represents the sum of two restriction fragments which originate from widely separated sites on the phage genome and thus any variation in the
58 AB1157 P
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POSITION ON T7 GE:NEITIC MAP
Fig, 5. Characterization of noninjectcd DNA isolated after infection of host cells by alkylated T7 phage. Upper panel: strain AB1157. Lower panel: strain BK2114. Noninj¢cted "~P-labelled phage DNA was hybridized to T7 I/pal restriction fragments ,is described in Materials ~md Methods, Results are expressed as the intensity of a given band for the noninjected DNA from alkylated T7 phage relative to the intensity of the corresl~)nding band for the noninjetted DNA from nonalkylated T7 phage. These relative intensities arc presented as a function of the l~sition of the restriction fragments on the genetic map of bacteriophage TT. Both alkylated and nonalkylated samples were hybridized to T7 Hpal restriction fragments which had been separated on the same gel and transferred onto the same nitrocellulose paper, which was subsequentlycut into strips for hybridization. Data are from one experiment in a set of 4, for strain ABI157, and 3, for strain BK2114. For these sets of experiments, the ratia of relative intensities of band E and band F+C+H was !.58±0.21 (a,e.: average error) and that of band B and band F+G + H was 2,82±0,67 (a.e.) for strain AB1157; the ratio of relative intensities of band E and band F + G + H was 2. I I ± 0,23 (a,e,) and that of band Band hand F+G+ H was 2,29+0.29 (a,e.) for strain BK2114. intensity of that band is difficult to interpret, The band representing fragment P, the smallest fragment analyzed, gave the weak,:st signal on all autoradiograms; its intensity,*was used only in cases where the signal was significan~ compared to that of the background.
addition, the ratios of bands present in the same proportion in noninjected DNA from alkylated or nonalkylated samples had values not far from 1. The first observation one can make from Fig. 5 is that both strains gave essentially the same pattern; bands E and B had higher intensities than bands from the left or right ends of the genome. In confirmation of the results in Table I, the data show that host-cell repair capacity does not influence T7 D N A injection. What is striking in Fig. 5 is the pattern of band intensities across the genome. As will now be demonstrated, it is this trend in ratios, rather than their absolute values, which can provide information on DNA injection. A horizontal line can be traced from band F + G and H to band O, at which point the intensity ratio begins to rise. Restriction fragments E, P and B, originating from positions just left of the L fragment, were clearly overrepresented in the noninjected DNA isolated from alkylated phage. Thus, alkylation by MMS appeared to physically block the injection of part of the phage genome, with the site of blockage located somewhat to the left of Hpal restriction fragment E. At the dose of 10 mM MMS used here, alkylation generally inactivated 98% of viable T7 phage particles as measured by plaque assay on cells proficient in repair of 3-methyladenine [20]. From the data in Fig. 5, we surmise that incomplete injection of phage DNA is certainly one cause of this inactivation. The intensity ratios of the band comprising L, M and N fragments, situated to the right of band B on the genetic map, were of comparable values to those of bands F + G and H through O. Thus, the bands from the right end of the genome did not appear to bc overrepresented in the noninjected DNA from alkylated phage. The explanation for this suprising observation was provided by direct examination of injection by nonalkylated phage.
Analysis of DNA injection by nonalkylated phage Analysis ~:~fDNA injection by alkylated bacteriophage T7 As sh,:wn in Table !, ~)NA injection was decreased by alkylation of phage, but the repair capacity of the host cell apreared to have no effect on injection. To further yetiS this point, we characterized the noninjetted D N A by hybridization. Fig, 5 presents a comparison of the DNA remaining uninjected at'ter infection of AB1157 or BK2114 cells by alkylated phage, Results are expressed as the ratio of the intensity of each restriction fragment band for alkylated phage as compared to that for nonaikylated phage, In each case, the nonalkylated and alkylated samples came from the same alkylation and injection experiment and hybridization of the noninjected D N A was carried out with restriction fragments which had originally been separated on the same agarose gel. These precautions reduced experimental variation. In
The data in Table 1 show that phage particles, prior to alkylation, were unable to totally inject their DNA into host cells. To further analyze this injection defect for nonalkylated phage, we compared hybridization patterns for nonalkylated phage after infection to those obtained for DNA extracted directly from purified phage particles, i.e., data shown in Fig. 3. The results, presented in Fig. 6, demonstrate a marked overrepresentation of the right three restriction fragments (bands L, M and N) in DNA isolated from capsids after infection of either strain by nonalkylated phage, as compared to T7 DNA. In general, restriction fragment N was the most increased in proportion followed by M and then L. The restriction fragment J, rightmost on the genetic map, was difficult to analyze because it comigrated with the I fragment which originates from the middle of the phage genome. Nonetheless, in these
59
experiments, the proportion of the I + J band was also increased compared to its proportion in phage DNA (data not shown). In addition, all Hpal restriction fragments were present in the noninjected DNA, suggesting that some phage particles were adsorbed to the host cell but were unable to inject any of their DNA. These observations indicate that populations of untreated phage particles were partially defective in DNA injection and that the rightmost segments of the genome, those which enter the cell last, were the most affected. Others have reported that some fraction of adsorbed T7 phage failed to participate in the infective cycle, presumably due to faulty injection [3,29]. However, this is the first time, to our knowledge, that an injection defect has been shown to specifically affect the last segments of the genome to enter the cell. These results provide an explanation for the unexpectedly low intensity ratios for bands L, M and N in patterns for alkylated phage (Fig. 5). Those bands were already greatly overrepresented in nonalkylated DNA compared to their proportion in the T7 genome; therefore little or no increase in the intensity ratio due to alkylation would be expected. For reasons explained above, band A was not considered in this analysis.
Effect of rifampicin on DNA injection by nonalkylated phage in establishing the experimental protocol for our initial injection experiments, we showed that rifampicin AB1157
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Fig. 6. Characterization of noninjected DNA isolated alter infection of host cells by nonalkylated T7 phage. Upper panel: strain AB1157. Lower panel: strain BK2114. Noninjected nonalkylated "~-'P-labelled phage DNA was hybridized to T7 Hpal restriction fragments as described in Materials and Methods. Results are expressed as the intensity of a given band for the noninjected DNA from nonalkylated T7 phage relative to the intensity (average intensity from four separate hybridizations) of the corresponding band in hybridizations carried out with purified 32P-labelled T7 DNA (see Fig. 3). These relative intensities are expressed as a functk)n of the position of the restriction fragments on the genetic map of bacteriophage T7. Data are from one experiment in a set of 8. for strain AB! 157, and t), for strain BK2114. For these sets of experiments, the ratio of relative intensities of band L + M + N and band F + G + H was 2.15+0.40 (a.e.) for strain AB1157 and 1.71 +0.28 (a.e.) for strain BK2114.
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Fig. 7. Characterization of noninjected DNA isolated after infection of rifampicin-treated host cells by nonaikylated bacteriophage TT. Upper panel: strain ABII57. Lower paneh strain BK2114. Noninjeered '~"P-labelled phage DNA was hybridized to T7 ttpal restriction fragments as described in Materials and Methods. Results are expressed its the intensity of a given band for noninjected DNA in the presence of rifinnpicin relative to the intensity of the corresponding band for noninjected DNA in the absence of rilampicin. These relative intensities are expressed as a function of the position of the restriction fragment on the genetic map of bacteriophage T7. Both sets of samples were hybridized to T7 Hpal restriction fragments which had been separated on the same gel and transferred onto the same nitrocellulose paper, which was subsequently cut into strips for hybridization. Data are from one experiment in a set of 4. for strain AB! 157, and 3, for strain BK2114.
significantly inhibited DNA injection. To further investigate this inhibition, we analyzed noninjected DNA from an injection experiment carried out in the presence of rifampicin. Fig. 7 shows that the intensity ratio between noninjected DNA in the presence and in the absence of rifampicin started to rise to the right of bands F + G and H. Other workers [3(i)] have also shown that only the leftmost end (about 1(1%, corresponding to fragment F) of T7 DNA entered cells when host RNA polymerase was inhibited by rifampicin. Since bands F and G were not separated in our experiments, we were not able to locate the point at which non-assisted injection stopped with the same precision as those authors, it is to be noted that the bands F + G and H never disappeared from our gels, as might be expected if all phages were able to inject at least the first part of their genome. An explanation for this is that some phages, although absorbed, injected no DNA whatsoever; these phages contributed their entire genomes to the hybridization patterns. Discussion
DNA b~jection by nonalkylated phage Many phages including T7 display an efficiency of plating (EOP) significantly lower than 1 [35]. The actual value of EOP represents the fraction of phage particles able to recognize and successfully infect ap-
0 propriate host cells. Following adsorption, DNA injection is the first step which must be completed for an infection to be productive. Our results (Fig. 6) show that untreated phage particles encountered difficulty in totally injecting their DNA into host cells under conditions we defined as physiological.Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome while others injected no DNA at all. For phages which had already adsorbed, problems at the stage of injection could explain why the EOP value is well below 1. To illustrate this, the data in Fig. 6 can be analyzed to estimate the relative fraction of phages which injected no DNA and that of those which injected all but the last 15% (fragments L, M, and N plus J which was not analyzed here). For this analysis, let us suppose that a horizontal line joins bands F + (3 and H and B. For both strains, the relative intensity of M, N, and L is roughly twice that of F + G, and H (and, by extension and neglecting scatter, all other fragments up to B). in molecular terms, this would mean that for eve~ DNA molecule which was not injected, there was one DNA molecule injected up to fragments M, N and L. Knowing that the injection defect was about 15% (Table I) and that fragments F, and and H represent the last 15% of the genome (Fig. 2), one can calculate that 13% of the phage particles injected no DNA and another 13% injected all but the final 15%. This simple analysis leads to an estimate of at least 26% of the phage particles which experienced injection problems. Our findings for untreated phage, as well as for alkylated phage, also provide direct physical evidence for unidirectional T7 DNA injection starting from the genetic left end of the phage genome. While Zavriev and Shemyakin [30] obtained similar physical evidence for this unidirectional injection, their host cells, pretreated with chloramphenicol, were in nonphysiological conditions at the time of phage infection.
DNA injection by alkylated phage DNA isolated from viral capsids after injection by aikTlated bacteriophage T7 displayed a marked enrichment of those sequences originating from positions between 60 and 85% from the left end of the T7 genetic map (Fig. 5); noninjected DNA from both alkTlated and nonalkylated phage were enriched in sequences beyond this point, i.e., from the extreme right end (Figs. 5 and 6). Under special conditions, the right-hand third of the T7 ('1"3) genome appears to interact with the phage capsid [32] and/or the cell wall [36]; these observations are probably pertinent to our results for DNA injection. During normal injection, phage particles can apparently overcome this special interaction; results in Fig. 6 show that, for nonalkylated phage, the only segments which were overrepresented in the nonin-
jected DNA fraction were those coming from the extreme right end (beyond the 85% position), in contrast, DNA injection by alkylated T7 phage was blocked at a site which left approximately one-third of the phage genome within the capsid. This suggests that alkylation somehow stabilized the binding of the right-hand third of the T7 genome to the phage capsid. This stabilization could be of a purely structural nature, involving simply a physical inhibition or a slowing down of the exit of DNA from the phage capsid. Such an idea finds support in the observation that molecules which bind to the minor groove blocked DNA translocation, as measured by in vitro encapsidation assays [37]. 3-Methyladenine, the toxic lesion which results from treatment of phage with MMS, is located in the minor groove and could thus hinder DNA translocation in our experiments. Alternatively or in combination with the above proposal, the stabilization of DNA-capsid interaction~ could be of a biochemical nature, in which case RNA polymerase activity would be so perturbed by alkylation adducts on the phage genome that transcription could not overcome the phage DNA-capsid interactions.
DNA injection in the presence of rifampicin The transcription-assisted model for T7 DNA entry into host cells is based principally on experimental results obtained in the presence of the antibiotic rifampicin, an inhibitor of E. coli RNA polymerase. Zavriev and Shemyakin [30] demonstrated that the antibiotic prevented entry of most of the T7 genome; Moffatt and Studier [3] showed that either E. coli RNA polymerase or T7 RNA polymerase, when supplied within the host cell by a plasmid which expresses T7 gene 1, permitted entry and expression of the '1"7 genome. DNA entry is thus normally coupled to transcription although nontranscription-assisted entry may occur under some conditions. Results presented here reinforce the idea that transcription is normally necessary for entry of the entire T7 genome. We also have shown that rifampicin inhibited DNA injection. We, as others [30], however, observed that some DNA entered the host cell in the presence of antibiotic; possibly there is a mode of DNA entry unlinked to transcription which permits DNA entry but no subsequent gene expression. In the absence of any antibiotic, a certain fraction of phage T7 particles experienced difficulty in injecting the right end of the genome (Fig. 6). Perhaps this hindered injection resulted from an uncoupling of the processes of injection and transcription. DNA injection and phage inactivation by MMS The observation that the biochemical characteristics of T7 development following alkylation varied significantly depending on the repair capacity of the host cell
61 (Fig. l and Refs. 14, 20)warrants some reflection on the mechanism of phage inactivation. An injection defect could conceivably be the ultimate cause of all phage inactivation observed when alkylated T7 phage infects a repair-proficient host; a genome lacking part of the right end of its sequence would be replicationdefective but were only a short segment to be missing, this defect would not be obvious until very late in the infective cycle when concatemers formed or assembly proteins gpl8 and gp 19 were needed. In contrast, phage inactivation was evident at a much earlier stage in the cycle in BK2114 cells, which are unable to remove 3-methyladenine from the phage DNA. Alkylated phage was unable to degrade host DNA (Fig. 11 through lack of expression of class I1 proteins [14]. Our attention thus far has been focussed on alkylated purines formed in phage DNA by MMS treatment. Other types of lesions, such as apurinic (AP) sites, are formed as secondary lesions after alkylation of phage. It is difficult to directly assess the contribution of AP sites to the injection defect we have observed here as the acid conditions required to introduce AP sites directly into the DNA of intact phage would undoubtedly affect the structure of the capsid proteins at the same time. However, incubation of alkylated phage for 24 h to induce depurination did decrease the amount of DNA injected [11]; it has been suggested that crosslinks between protein and DNA, formed via AP sites, might be responsible for this [38]. For the experiments involving alkylated phage reported here, it appears unlikely that such capsid-protein crosslinks could account for the observed injection defect; the number of AP sites in alkylated phage is small compared to the total number of base alkylations [39] so that the likelihood of crosslinking DNA to capsid proteins would be very slight. Furthermore, if T7 DNA is arranged within the capsid as is the DNA of other double-stranded DNA phages, e.g., T4 and lambda [40,41], then the DNA last to be encapsidated should be located on the outside of the DNA mass, in close contact with the capsid proteins; with this model, one would thus expect that any protein-DNA crosslinks would be formed within the left end of the T7 genome. Our results show that alkylated phage injected its left end just as efficiently as nonalkylated phage (Fig. 5). This makes it unlikely that protein-DNA crosslinks contribute to the injection defect caused by MMS.
Repair capacity and T7 DNA injection Starting from the proposed transcription-assisted model for T7 DNA injection [1-3] and reasoning that alkylation-induced changes in phage DNA could affect its transcription, we proposed that a further decrease in injection, caused by the inability of RNA polymerase to transcribe DNA containing unrepaired 3-methyladenines, could explain the major decrease in host-cell
reactivation, observed when alkylated phage infected the repair-defective strain BK2114 as compared to the repair-proficient strain AB1157. AIkylation of bacteriophage T7 indeed caused an injection defect, but that defect was the same for both strains (Table I). Physical characterization of noninjected DNA from alkylated phage clearly showed that the same sequences were injected into both strains (Fig. 5). We therefore conclude that DNA injection is independent of the hostcell's capacity for repair of 3-methyladenine residues. Since our experiments measured the end point of DNA injection, they of course do not rule out a difference in the kinetics of DNA entry. If repair does not affect injection, how then does damaged DNA succeed in entering the host cell? It seems likely that entry of alkylated DNA depends upon E. coli RNA polymerase alone since this enzyme is relatively insensitive to alkylation lesions compared to its phage counterpart, T7 RNA polymerase [42]. The bacterial enzyme, by reading through the termination signal at the end of the early region, could transcribe the entire late region and thus mediate DNA entry. Just how the host enzyme would deal with other types of DNA damage, such as pyrimidine dimers, known to inhibit transcription in vitro [43], to bring the DNA into the cell is less clear. Ult;aviolet-damaged phage DNA does actually enter the host cell where it is repaired; this host-cell reactivation leads to increased phage survival [44]. Direct analysis of DNA injection by methods used here should help to understand how phage DNA carrying lesions other than alkyl groups enters the host cell and thus shed new light on the injection process.
Acknowledgments We are grateful to B. Karska-Wysocki, J.-F. Racine and G. Sanchez for critical comments on the manuscript. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.
References 1 Zavriev, S.K. and Shemyakin, M.F. (1981) FEBS Lel[. 131, 99102. 2 McAllister, W.T., Morris, C., Rosenberg, A.H. and Studier. F.W. (1981) J. IV[ol. Biol. 153, 527-544. 3 Moffatt, B. and Studier, F.W. (1988) J. Bacteriol. 170, 2(195-2105. 4 Pao, C.-C. and Speyer, J.F. (1973)J. Virol. 11, 1024-1026. 5 Karska-Wysocki, B., Mamet-Bratley, M.D. and Verly, W.G. (1976) J. Virol. 19, 318-324. 6 Karska-Wysocki, B., Mamel Bratley, M.D. and Przewlocki. G. (19771 J. Virol. 24, 716-719. 7 Mamet-Bratley, M.D. (1971)Biochim. Biophys. Acta 247. 233242. 8 Nath, S.T., Lee, M.-S. and Romano, L. (19871 Nucleic Acids Reso 15, 4257-4271.
62 9 Shi, Y,-B., Gamper, H. and Hearst, J.E, (1987) Nucleic Acids Res. 15, 6843-6854. 10 Shi, Y.-EL, Gamper, H. and Hearst, ,I,E. (1988) J. Biol. Chem. 263, 527-534. !1 Karska-Wysocki, EL, Thibodeau, L. and Verly, W.G. (1976) Biochim. Biophys. Acta 435, 184-191. 12 MamebBratley, M.D., Zollinger, M. and Karska-Wysocki, B. (1982) Can, J, Biochem. 60, 232-242. 13 Mamet-Bratley, M,D., Czaika, G,, Racine, J.-F, and KarskaWysocki, EL (1988) J. Ceil. Biochem, Suppl. 12A, 319. 14 Czaika, G., Racine, £-F, and Mamet-Bratley, M.D. (1988) Mol. Gen. (Life Sci, Adv,) 7, 179-185, 15 Bachmann, BJ. (1987) in Escherichia coil and Salmonella ty. phimurium, Cellular and Molecular Biology (Neidhardt, F.C, ed,), VoL 2, pp, 1190-1219, American Society for Microbiology, Washington, 16 Evensen, G. and Seeberg, E. (1982) Nature 296, 773-775. 17 Thomas, C,A., Jr, and Abelson, J. (1966) in Procedures in Nucleic Acid Research (Cantoni, G,L. and Davies, D.R,, eds.), Vol. 1, pp. 553-561, Harper and Row Publishers, New York. 18 Franklin, W,A. and Haseltine, W,A, (1984) Proc. Natl. Acad. Sci. USA 81, 3821-3824. 19 Mamet-Bratley, M.D. and Karska-Wysocki, B. (1982) Biochim. Biophys, Acta 698, 29-34. 20 Czaika, G , Mamet-Bratley, M.D. and Karska-Wysocki, B. (1986) Met, Res, 166, 1-8, 21 Adams, M,H, (1959) Bacteriophages, pp. 450-451, Interscience Publishers, New York, 22 Langman, L. and Paetkau, V. (1978) J. Virol. 25, 562-569. 23 Paetkau, V. and Langman, L. (1975) Anal. Biochem. 65, 525-532. 24 Schleif, R.F. and Wensink, P.C. (1981) Practical Methods in Molecular Biology, pp. 152-156, Springer-Verlag, New York. 25 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, p. 85, Cold Spring Harbor Laboratoty Press, Cold Spring Harbor.
26 Denhardt, D,T. (1966) Biochem. Biophys. Res. Commun. 23, 641-646. 27 Dunn, JJ. and Studier, F.W. (1983) J. Mol. Biol. 166, 477-535. 28 Hartman, P.S., Eisenstark, A. and Pauw, P.G. (1979) Proc. Natl. Acad. Sci. USA 76, 3228-3232. 29 Benbasat, J A., Burck, K.B, and Miller, R.C., Jr. (1978) Virology 87, 164-171. 30 Zavriev, S.K. and Shemyakin, M.F. (1982) Nucleic Acids Res. 10, 1635-1652, 31 Bjornsti, M.-A., Reiily, B.E. and Anderson, D.L. (1983) J. Virol. 45, 383-396. 32 Shibata, H,, Fujisawa, H. and Minagawa, T. (1987) J. Mol. Biol. 196, 845-851, 33 Rosenberg, A.H., Simon, M.N., Studier, F.W. and Roberts, RJ. (1979) J, Mol, Biol, 135, 907-915. 34 McDonnell, M.W., Simon, M.N. and Studier, F.W. (1977)J. Mol. Biol. 110, 119-146, 35 Goldberg, E. (1983) in Bacteriophage T4 (Mathews, C.K., Kutter, E.M., Mosig, G. and Berget, P.B., eds.), pp, 32-39, American Society for Microbiology, Washington, 36 Zavriev, S.K. and Vorob'ev, S.M. (1984) FEBS Lett. 165, 31-34. 37 Fujisawa, H., Hamada, K., Shibata, H. and Minagawa, T. (1987) Virology 161,228-233. 38 Flam~e, P,-A. and Verly, W.G. (1985) Biochem. J. 299, 173-181. 39 Verly, W.G., Crine, P., Bannon, P. and Forget, A, (1974) Biochim. Biophys. Acta 34% 204-213. 40 Black, L.W., Newcomb, W.W., Boring, J.W. and Brown, J.C. (1985) Proc. Natl. Acad. Sci. USA 82, 7960-7964. 41 Brown, J.C. and Newcomb, W.W. (1986) J. Virol. 60, 564-568. 42 Racine, J.-F. and Mamet-Bratley, M.D. (1990) Proc. Can. Fed. Biol. Soc. 33, 66. 43 Sauerbier, W., MiUette, R.L. and Hackett, P.B., Jr, (1970) Biochim. Biophys. Acta 209, 368-386. 44 Kuemmerle, N.B. and Masker, W.E. (1977) J. Viroi. 23, 509-516.
Biocidmica et Biophysica Acta, ! 130(1992) 52-62 i.-
z
A ......
Ld 01
Z
LMNJ
15
,J I--
01 O~
"r I-0
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v
-
KL M
Fig. 3. Densitometric scan of an autoradiogram which represents ~zP-labelled T7 D N A hybridized to T 7 / / p a l restriction fragments separated by electrophoresis on a !.2% agarose gel. Bacteriophage T7 containing "~'P-labellcd D N A was extensively dialyzed against 10 mM NaCI, 50 mM Tris-HCI (pH 8.0), 10 mM MgC!-, and the phage
DNA was purified and hybridized to T7 Hpal restriction fragments as described in Materials and Methods for the analysis of noninjeered DNA. The photograph of the autoradiogram was intentionally overexposed for publication.
INTENSITY (%)
Fig. 4. Quantitative analysis of hybridization between llpal T7 restriction fragments and T7 DNA. The relative intensity (~,:) of each band was compared to the expected relative intensity calculated from the molecular length of the corresponding ttpai u'estriction fragment [34]. Experimental intensities were obtained by averaging the results of four separate hybridization experiments. Average errors are indicated by the vertical lines while the broken diagonal line rcprescnt~ perfect confi~rmity of experimental and theoretical values.
indicating that extraction procedures and hybridization conditions permitted equivalent quantitative recovery of all parts of the genome. The intensity of band A was somewhat underestimated by our method. We believe this to be due to the fact that restriction fragment A, by far the largest of the Hpal products, did not elute from the agarose gel as fast as the other smaller fragments. By prolonging the transfer period (already about 15 h) we would have run the risk of losing the smallest fragments by elution out of and beyond the nitrocellulose sheet. Band A is thus very sensitive to transfer conditions and its intensity was considered significant only for hybridizations relating to the same initial gel. Calculation of the intensity of band E was also difficult because it is seen only as a shoulder of the F + G band in densitometric scans. In our analysis, we have combined the intensities of certain bands: L, M and N, C and D, and F + G and H (see Fig. 2). The first three correspond to three adjacent Hpal restriction fragments from the right end (last part to be injected) of the phage genome, while the F + G and H bands correspond to adjacent fragments from the left (first to be injected) end. The C and D bands correspond to adjacent restriction fragments situated just to the right of G on the Hpal restriction map of T7. By combining these bands, we have decreased the average error involved in the computer-determined attribution of peak-widths: M and N, C and D, as well as F + G and H are neighboring bands in the electrophoretic patterns and hence, contiguous peaks in the densitometric profiles. Unfortunately, band 1 + J represents the sum of two restriction fragments which originate from widely separated sites on the phage genome and thus any variation in the
58 AB1157 P
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100
POSITION ON T7 GE:NEITIC MAP
Fig, 5. Characterization of noninjectcd DNA isolated after infection of host cells by alkylated T7 phage. Upper panel: strain AB1157. Lower panel: strain BK2114. Noninj¢cted "~P-labelled phage DNA was hybridized to T7 I/pal restriction fragments ,is described in Materials ~md Methods, Results are expressed as the intensity of a given band for the noninjected DNA from alkylated T7 phage relative to the intensity of the corresl~)nding band for the noninjetted DNA from nonalkylated T7 phage. These relative intensities arc presented as a function of the l~sition of the restriction fragments on the genetic map of bacteriophage TT. Both alkylated and nonalkylated samples were hybridized to T7 Hpal restriction fragments which had been separated on the same gel and transferred onto the same nitrocellulose paper, which was subsequentlycut into strips for hybridization. Data are from one experiment in a set of 4, for strain ABI157, and 3, for strain BK2114. For these sets of experiments, the ratia of relative intensities of band E and band F+C+H was !.58±0.21 (a,e.: average error) and that of band B and band F+G + H was 2,82±0,67 (a.e.) for strain AB1157; the ratio of relative intensities of band E and band F + G + H was 2. I I ± 0,23 (a,e,) and that of band Band hand F+G+ H was 2,29+0.29 (a,e.) for strain BK2114. intensity of that band is difficult to interpret, The band representing fragment P, the smallest fragment analyzed, gave the weak,:st signal on all autoradiograms; its intensity,*was used only in cases where the signal was significan~ compared to that of the background.
addition, the ratios of bands present in the same proportion in noninjected DNA from alkylated or nonalkylated samples had values not far from 1. The first observation one can make from Fig. 5 is that both strains gave essentially the same pattern; bands E and B had higher intensities than bands from the left or right ends of the genome. In confirmation of the results in Table I, the data show that host-cell repair capacity does not influence T7 D N A injection. What is striking in Fig. 5 is the pattern of band intensities across the genome. As will now be demonstrated, it is this trend in ratios, rather than their absolute values, which can provide information on DNA injection. A horizontal line can be traced from band F + G and H to band O, at which point the intensity ratio begins to rise. Restriction fragments E, P and B, originating from positions just left of the L fragment, were clearly overrepresented in the noninjected DNA isolated from alkylated phage. Thus, alkylation by MMS appeared to physically block the injection of part of the phage genome, with the site of blockage located somewhat to the left of Hpal restriction fragment E. At the dose of 10 mM MMS used here, alkylation generally inactivated 98% of viable T7 phage particles as measured by plaque assay on cells proficient in repair of 3-methyladenine [20]. From the data in Fig. 5, we surmise that incomplete injection of phage DNA is certainly one cause of this inactivation. The intensity ratios of the band comprising L, M and N fragments, situated to the right of band B on the genetic map, were of comparable values to those of bands F + G and H through O. Thus, the bands from the right end of the genome did not appear to bc overrepresented in the noninjected DNA from alkylated phage. The explanation for this suprising observation was provided by direct examination of injection by nonalkylated phage.
Analysis of DNA injection by nonalkylated phage Analysis ~:~fDNA injection by alkylated bacteriophage T7 As sh,:wn in Table !, ~)NA injection was decreased by alkylation of phage, but the repair capacity of the host cell apreared to have no effect on injection. To further yetiS this point, we characterized the noninjetted D N A by hybridization. Fig, 5 presents a comparison of the DNA remaining uninjected at'ter infection of AB1157 or BK2114 cells by alkylated phage, Results are expressed as the ratio of the intensity of each restriction fragment band for alkylated phage as compared to that for nonaikylated phage, In each case, the nonalkylated and alkylated samples came from the same alkylation and injection experiment and hybridization of the noninjected D N A was carried out with restriction fragments which had originally been separated on the same agarose gel. These precautions reduced experimental variation. In
The data in Table 1 show that phage particles, prior to alkylation, were unable to totally inject their DNA into host cells. To further analyze this injection defect for nonalkylated phage, we compared hybridization patterns for nonalkylated phage after infection to those obtained for DNA extracted directly from purified phage particles, i.e., data shown in Fig. 3. The results, presented in Fig. 6, demonstrate a marked overrepresentation of the right three restriction fragments (bands L, M and N) in DNA isolated from capsids after infection of either strain by nonalkylated phage, as compared to T7 DNA. In general, restriction fragment N was the most increased in proportion followed by M and then L. The restriction fragment J, rightmost on the genetic map, was difficult to analyze because it comigrated with the I fragment which originates from the middle of the phage genome. Nonetheless, in these
59
experiments, the proportion of the I + J band was also increased compared to its proportion in phage DNA (data not shown). In addition, all Hpal restriction fragments were present in the noninjected DNA, suggesting that some phage particles were adsorbed to the host cell but were unable to inject any of their DNA. These observations indicate that populations of untreated phage particles were partially defective in DNA injection and that the rightmost segments of the genome, those which enter the cell last, were the most affected. Others have reported that some fraction of adsorbed T7 phage failed to participate in the infective cycle, presumably due to faulty injection [3,29]. However, this is the first time, to our knowledge, that an injection defect has been shown to specifically affect the last segments of the genome to enter the cell. These results provide an explanation for the unexpectedly low intensity ratios for bands L, M and N in patterns for alkylated phage (Fig. 5). Those bands were already greatly overrepresented in nonalkylated DNA compared to their proportion in the T7 genome; therefore little or no increase in the intensity ratio due to alkylation would be expected. For reasons explained above, band A was not considered in this analysis.
Effect of rifampicin on DNA injection by nonalkylated phage in establishing the experimental protocol for our initial injection experiments, we showed that rifampicin AB1157
L+M+N
P
>F+G+H
I.-
(/) z
C+D
K
2
B
A
LIJ I--
E
z
3K2114
IM
_>
L+M+N £
2 .,J
I.iJ a,,
:+G+H 1
B
K m
C+D
O &
o
Too POSITION ON T7 GENETIC MAP
Fig. 6. Characterization of noninjected DNA isolated alter infection of host cells by nonalkylated T7 phage. Upper panel: strain AB1157. Lower panel: strain BK2114. Noninjected nonalkylated "~-'P-labelled phage DNA was hybridized to T7 Hpal restriction fragments as described in Materials and Methods. Results are expressed as the intensity of a given band for the noninjected DNA from nonalkylated T7 phage relative to the intensity (average intensity from four separate hybridizations) of the corresponding band in hybridizations carried out with purified 32P-labelled T7 DNA (see Fig. 3). These relative intensities are expressed as a functk)n of the position of the restriction fragments on the genetic map of bacteriophage T7. Data are from one experiment in a set of 8. for strain AB! 157, and t), for strain BK2114. For these sets of experiments, the ratio of relative intensities of band L + M + N and band F + G + H was 2.15+0.40 (a.e.) for strain AB1157 and 1.71 +0.28 (a.e.) for strain BK2114.
AB1157
A E
C+O
B K F+G+H
W
>
L+M'fN
I
BK2114
3
o
I--
< _1 ILl t~
2
A
B
C+D
L÷M+N
F+G+H
E K
i
o
50 POSITION
ON T 7 G E N E T I C
100 MAP
Fig. 7. Characterization of noninjected DNA isolated after infection of rifampicin-treated host cells by nonaikylated bacteriophage TT. Upper panel: strain ABII57. Lower paneh strain BK2114. Noninjeered '~"P-labelled phage DNA was hybridized to T7 ttpal restriction fragments as described in Materials and Methods. Results are expressed its the intensity of a given band for noninjected DNA in the presence of rifinnpicin relative to the intensity of the corresponding band for noninjected DNA in the absence of rilampicin. These relative intensities are expressed as a function of the position of the restriction fragment on the genetic map of bacteriophage T7. Both sets of samples were hybridized to T7 Hpal restriction fragments which had been separated on the same gel and transferred onto the same nitrocellulose paper, which was subsequently cut into strips for hybridization. Data are from one experiment in a set of 4. for strain AB! 157, and 3, for strain BK2114.
significantly inhibited DNA injection. To further investigate this inhibition, we analyzed noninjected DNA from an injection experiment carried out in the presence of rifampicin. Fig. 7 shows that the intensity ratio between noninjected DNA in the presence and in the absence of rifampicin started to rise to the right of bands F + G and H. Other workers [3(i)] have also shown that only the leftmost end (about 1(1%, corresponding to fragment F) of T7 DNA entered cells when host RNA polymerase was inhibited by rifampicin. Since bands F and G were not separated in our experiments, we were not able to locate the point at which non-assisted injection stopped with the same precision as those authors, it is to be noted that the bands F + G and H never disappeared from our gels, as might be expected if all phages were able to inject at least the first part of their genome. An explanation for this is that some phages, although absorbed, injected no DNA whatsoever; these phages contributed their entire genomes to the hybridization patterns. Discussion
DNA b~jection by nonalkylated phage Many phages including T7 display an efficiency of plating (EOP) significantly lower than 1 [35]. The actual value of EOP represents the fraction of phage particles able to recognize and successfully infect ap-
0 propriate host cells. Following adsorption, DNA injection is the first step which must be completed for an infection to be productive. Our results (Fig. 6) show that untreated phage particles encountered difficulty in totally injecting their DNA into host cells under conditions we defined as physiological.Some injected all their DNA except those segments located in the rightmost 15% of the T7 genome while others injected no DNA at all. For phages which had already adsorbed, problems at the stage of injection could explain why the EOP value is well below 1. To illustrate this, the data in Fig. 6 can be analyzed to estimate the relative fraction of phages which injected no DNA and that of those which injected all but the last 15% (fragments L, M, and N plus J which was not analyzed here). For this analysis, let us suppose that a horizontal line joins bands F + (3 and H and B. For both strains, the relative intensity of M, N, and L is roughly twice that of F + G, and H (and, by extension and neglecting scatter, all other fragments up to B). in molecular terms, this would mean that for eve~ DNA molecule which was not injected, there was one DNA molecule injected up to fragments M, N and L. Knowing that the injection defect was about 15% (Table I) and that fragments F, and and H represent the last 15% of the genome (Fig. 2), one can calculate that 13% of the phage particles injected no DNA and another 13% injected all but the final 15%. This simple analysis leads to an estimate of at least 26% of the phage particles which experienced injection problems. Our findings for untreated phage, as well as for alkylated phage, also provide direct physical evidence for unidirectional T7 DNA injection starting from the genetic left end of the phage genome. While Zavriev and Shemyakin [30] obtained similar physical evidence for this unidirectional injection, their host cells, pretreated with chloramphenicol, were in nonphysiological conditions at the time of phage infection.
DNA injection by alkylated phage DNA isolated from viral capsids after injection by aikTlated bacteriophage T7 displayed a marked enrichment of those sequences originating from positions between 60 and 85% from the left end of the T7 genetic map (Fig. 5); noninjected DNA from both alkTlated and nonalkylated phage were enriched in sequences beyond this point, i.e., from the extreme right end (Figs. 5 and 6). Under special conditions, the right-hand third of the T7 ('1"3) genome appears to interact with the phage capsid [32] and/or the cell wall [36]; these observations are probably pertinent to our results for DNA injection. During normal injection, phage particles can apparently overcome this special interaction; results in Fig. 6 show that, for nonalkylated phage, the only segments which were overrepresented in the nonin-
jected DNA fraction were those coming from the extreme right end (beyond the 85% position), in contrast, DNA injection by alkylated T7 phage was blocked at a site which left approximately one-third of the phage genome within the capsid. This suggests that alkylation somehow stabilized the binding of the right-hand third of the T7 genome to the phage capsid. This stabilization could be of a purely structural nature, involving simply a physical inhibition or a slowing down of the exit of DNA from the phage capsid. Such an idea finds support in the observation that molecules which bind to the minor groove blocked DNA translocation, as measured by in vitro encapsidation assays [37]. 3-Methyladenine, the toxic lesion which results from treatment of phage with MMS, is located in the minor groove and could thus hinder DNA translocation in our experiments. Alternatively or in combination with the above proposal, the stabilization of DNA-capsid interaction~ could be of a biochemical nature, in which case RNA polymerase activity would be so perturbed by alkylation adducts on the phage genome that transcription could not overcome the phage DNA-capsid interactions.
DNA injection in the presence of rifampicin The transcription-assisted model for T7 DNA entry into host cells is based principally on experimental results obtained in the presence of the antibiotic rifampicin, an inhibitor of E. coli RNA polymerase. Zavriev and Shemyakin [30] demonstrated that the antibiotic prevented entry of most of the T7 genome; Moffatt and Studier [3] showed that either E. coli RNA polymerase or T7 RNA polymerase, when supplied within the host cell by a plasmid which expresses T7 gene 1, permitted entry and expression of the '1"7 genome. DNA entry is thus normally coupled to transcription although nontranscription-assisted entry may occur under some conditions. Results presented here reinforce the idea that transcription is normally necessary for entry of the entire T7 genome. We also have shown that rifampicin inhibited DNA injection. We, as others [30], however, observed that some DNA entered the host cell in the presence of antibiotic; possibly there is a mode of DNA entry unlinked to transcription which permits DNA entry but no subsequent gene expression. In the absence of any antibiotic, a certain fraction of phage T7 particles experienced difficulty in injecting the right end of the genome (Fig. 6). Perhaps this hindered injection resulted from an uncoupling of the processes of injection and transcription. DNA injection and phage inactivation by MMS The observation that the biochemical characteristics of T7 development following alkylation varied significantly depending on the repair capacity of the host cell
61 (Fig. l and Refs. 14, 20)warrants some reflection on the mechanism of phage inactivation. An injection defect could conceivably be the ultimate cause of all phage inactivation observed when alkylated T7 phage infects a repair-proficient host; a genome lacking part of the right end of its sequence would be replicationdefective but were only a short segment to be missing, this defect would not be obvious until very late in the infective cycle when concatemers formed or assembly proteins gpl8 and gp 19 were needed. In contrast, phage inactivation was evident at a much earlier stage in the cycle in BK2114 cells, which are unable to remove 3-methyladenine from the phage DNA. Alkylated phage was unable to degrade host DNA (Fig. 11 through lack of expression of class I1 proteins [14]. Our attention thus far has been focussed on alkylated purines formed in phage DNA by MMS treatment. Other types of lesions, such as apurinic (AP) sites, are formed as secondary lesions after alkylation of phage. It is difficult to directly assess the contribution of AP sites to the injection defect we have observed here as the acid conditions required to introduce AP sites directly into the DNA of intact phage would undoubtedly affect the structure of the capsid proteins at the same time. However, incubation of alkylated phage for 24 h to induce depurination did decrease the amount of DNA injected [11]; it has been suggested that crosslinks between protein and DNA, formed via AP sites, might be responsible for this [38]. For the experiments involving alkylated phage reported here, it appears unlikely that such capsid-protein crosslinks could account for the observed injection defect; the number of AP sites in alkylated phage is small compared to the total number of base alkylations [39] so that the likelihood of crosslinking DNA to capsid proteins would be very slight. Furthermore, if T7 DNA is arranged within the capsid as is the DNA of other double-stranded DNA phages, e.g., T4 and lambda [40,41], then the DNA last to be encapsidated should be located on the outside of the DNA mass, in close contact with the capsid proteins; with this model, one would thus expect that any protein-DNA crosslinks would be formed within the left end of the T7 genome. Our results show that alkylated phage injected its left end just as efficiently as nonalkylated phage (Fig. 5). This makes it unlikely that protein-DNA crosslinks contribute to the injection defect caused by MMS.
Repair capacity and T7 DNA injection Starting from the proposed transcription-assisted model for T7 DNA injection [1-3] and reasoning that alkylation-induced changes in phage DNA could affect its transcription, we proposed that a further decrease in injection, caused by the inability of RNA polymerase to transcribe DNA containing unrepaired 3-methyladenines, could explain the major decrease in host-cell
reactivation, observed when alkylated phage infected the repair-defective strain BK2114 as compared to the repair-proficient strain AB1157. AIkylation of bacteriophage T7 indeed caused an injection defect, but that defect was the same for both strains (Table I). Physical characterization of noninjected DNA from alkylated phage clearly showed that the same sequences were injected into both strains (Fig. 5). We therefore conclude that DNA injection is independent of the hostcell's capacity for repair of 3-methyladenine residues. Since our experiments measured the end point of DNA injection, they of course do not rule out a difference in the kinetics of DNA entry. If repair does not affect injection, how then does damaged DNA succeed in entering the host cell? It seems likely that entry of alkylated DNA depends upon E. coli RNA polymerase alone since this enzyme is relatively insensitive to alkylation lesions compared to its phage counterpart, T7 RNA polymerase [42]. The bacterial enzyme, by reading through the termination signal at the end of the early region, could transcribe the entire late region and thus mediate DNA entry. Just how the host enzyme would deal with other types of DNA damage, such as pyrimidine dimers, known to inhibit transcription in vitro [43], to bring the DNA into the cell is less clear. Ult;aviolet-damaged phage DNA does actually enter the host cell where it is repaired; this host-cell reactivation leads to increased phage survival [44]. Direct analysis of DNA injection by methods used here should help to understand how phage DNA carrying lesions other than alkyl groups enters the host cell and thus shed new light on the injection process.
Acknowledgments We are grateful to B. Karska-Wysocki, J.-F. Racine and G. Sanchez for critical comments on the manuscript. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.
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