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Concatemers in a rapidly sedimenting, replicating bacteriophage T7 DNA
VIROLOGY
123,4?4-479
Concatemers
(1982)
in a Rapidly Sedimenting,
Replicating
Bacteriophage
T7 DNA
PHILIP SERWER, GISELE A. GREENHAW, AND JERRY L. ALLEN Department
of Biochemistry,
The University
of Texas Health Science Center, San Antonio,
Texas 78284
Received August 2, 1982; accepted September 14, 1982 In previous studies, replicating bacteriophage T7 DNA was isolated as a rapidly sedimenting, multigenome-sized complex (100 S’ DNA), consisting of double-stranded DNA with some single-stranded regions. In the present study, 100 Sf DNA was digested with nuclease Sl and the products were fractionated by velocity sedimentation and analyzed by electron microscopy and digestion with restriction enzymes, followed by agarose gel electrophoresis. Some of the fragments released from 100 S+ DNA by nuclease Sl are linear, heterogeneous in length, and one to five times as long as mature T7 DNA. Uniformlength DNA’s one, two, and four times the length of mature T7 DNA were also present in sufficiently large amounts to form visible bands during velocity sedimentation.
During their assembly in uiuo, several bacteriophages with duplex DNA, including T4, T7, X, and P22, have been found to produce postreplicative molecules of DNA that are end-to-end aggregates of the mature bacteriophage DNA and that are referred to as concatemers (reviewed in (1-5). Concatemers produced by bacteriophage T7 have been fractionated by velocity sedimentation in sucrose gradients (6), and it was reported that these concatemers have single-stranded regions at the termini of some of their constituent mature genomes (7). Because of this observation and the observation that T7 DNA is linear during the first few rounds of replication (8, 9), joining of linear replication products by hydrogen bonding at single-stranded termini has been proposed to be the mechanism of formation of T7 concatemers (7, 10). However, subsequently a more random distribution of single-stranded regions in T7 concatemers has been proposed (12). If the latter study is correct, there is no direct evidence of terminal joining during the formation of T7 concatemers. Evidence has been presented indicating that T7 concatemers are packaged in a preformed T7 capsid and after packaging are cut to mature size (12, 13). Subsequent to the appearance of the linear replicative forms of T7 DNA described 0042-6822/82/160474-06$02.00/O Copyright All rights
0 19EZ by Academic Press, Inc. of reproduction in any form reserved.
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in (8,9), replicating T7 DNA becomes part of a larger complex of DNA sedimenting at rates greater than 100 S and referred to as 100 S DNA (14); a more deproteinized form of 100 S+ DNA has been referred to as flowers (15). Digestion of 100 S DNA with nucleases specific for single-stranded DNA releases DNA sedimenting as, but not yet demonstrated to be, T7 concatemers (II, 14). As emphasized in (II), this DNA sediments as though it were heterogeneous in length, and, therefore, does not provide evidence of the terminal joining previously discussed (10). To further analyze fragments of 100 S+ T7 DNA, procedures of agarose gel electrophoresis capable of resolving T7 concatemers (16) were subsequently used to fractionate fragments of 100 S DNA released by nuclease Sl, specific for singlestranded DNA. It was found, in agreement with data previously obtained (11,14), that the mobility of most concatemer-migrating (and more slowly migrating) DNA released varied continuously. However, in contrast to the previous studies, it was found that a comparatively small amount of released DNA formed sharp bands at the positions of mature-length T7 DNA (monomeric DNA) and dimeric concatemers (17). If the concatemer-migrating DNA in (I 7) were really concatemeric,
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in the present study fragments of 100 S+ DNA sedimenting as concatemers have been characterized. In addition, the sedimentation profile of this DNA has been observed at a resolution higher than in previous studies. To fractionate and observe fragments of 100 S+ T7 DNA produced by digestion to completion with nuclease Sl, these fragments were sedimented in sucrose gradients containing 1 pg/ml ethidium bromide (ethidium bromide-sucrose gradients) and photographs of the long-wave ultraviolet light-induced fluorescence in the gradients were taken (18). As previously found using agarose gel electrophoresis (17), all regions of ethidium bromidesucrose gradients potentially containing concatemers isolated at 22 min after in-
these results would provide evidence of the joining by single DNA strands of monomeric DNA’s within concatemers, possibly the terminal joining previously discussed (10). The observation of concatemer-migrating DNA in (17) was accomplished by staining (without fractionating) gels, a procedure which facilitates the detection of faint bands; this procedure has not yet been used for the detection of fragments of 100 S DNA after sedimentation, although it is possible to do so (18). The concatemer-migrating fragments of 100 S+ DNA were not rigorously shown to be concatemeric in any of the above studies. Branched or circular DNA’s may migrate with concatemers during velocity sedimentation (19) and possibly also during agarose gel electrophoresis. Therefore,
menliscus -
-meniscus
-
-monomer -dimer -tetramer IT4
T7
T5 T4 -
a
b
c
FIG. 1. Sedimentation of nuclease Sl-fragmented 100 S+ T7 DNA. A loo-ml culture of E. coli BB/ 1 was infected with bacteriophage T7; 40-ml portions of this culture were chilled at 22 and 25 min after infection and 100 S+ DNA was prepared from both after lysis with Sarkosyl, as previously described (17). A 60-~1, undialyzed portion of both 100 S DNA preparations, containing DNA from 1.4 X 10’ cells, was digested with 100 units/ml nuclease Sl (Sigma) as previously described (17). After dialysis against 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 M EDTA for 16 hr and addition of 0.25 pg of bacteriophage T4 DNA (18), the digested DNA’s were sedimented through ethidium bromide-sucrose gradients in the above buffer, followed by photography of fluorescence, as previously described (18). Also sedimented was a sample containing 0.40 pg each of DNA’s from bacteriophages T4, T5, and 0.25 pg of DNA from bacteriophage T7 (18). (a) T4, T5, and T7 DNA, (b) 100 S+ DNA digest, 22 min; (c) 106 S+ DNA digest, 25 min. The dependence of distance sedimented in ethidium bromide-sucrose gradients on the M, of duplex DNA was previously established using the mature DNA’s of bacteriophages T4, T5, and T7 as standards (18). However, T4 DNA is glucosylated; T5 and T7 DNA’s are not. In (18), correction for the effect of glucosylation of T4 DNA was not made. When this is done, the exponent in Eq. 3 of Ref. (18) is changed to 0.36; this revised exponent was used for all calculations made here. Unglucosylated T4 DNA (26) sedimented 7 f 1% slower than glucosylated T4 DNA.
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fection (Fig. lb) and at 25 min after infection (Fig. lc) were more fluorescent than the background regions of a gradient containing a mixture of mature DNA’s from bacteriophages T4, T5, and T7 (Fig. la). This indicates that molecules too variable in sedimentation rate to form sharp bands are present in the gradients of Figs. lb, c. However, superimposed on this continuously varying background were three bands closer to the origin of sedimentation than T4 DNA added to the samples in Figs. lb, c as an internal standard for molecular weight (A4,) (by sedimentation in a separate gradient without the T4 DNA, it was shown that no band formed by fragments of 100 S+ DNA is at the position of the T4 DNA standard). Correcting the position of T4 DNA for glucosylation of this DNA (legend to Fig. l), the positions of the bands formed by fragments of 100 S+ DNA are calculated (legend to Fig. 1) to be the positions of linear, duplex DNA molecules with MZ’s that are 0.93 +- 0.1 (least rapidly sedimenting), 2.0 + 0.1, and 4.2 + 0.2 (most rapidly sedimenting) times the M, of mature T7 DNA. These ratios are not significantly different from 1, 2, and 4; evidence is presented below suggesting that DNA’s forming the bands are T7 concatemers. Therefore, DNA’s forming the two bands furthest from the origin of sedimentation will be referred to as dimeric concatemers and tetrameric concatemers, respectively; DNA forming the band closest to the origin of sedimentation will be referred to as monomeric DNA. A possible interpretation of the absence of a band formed by “trimeric concatemers” is discussed below. It has previously been shown that restriction enzyme digestion of unfractionated DNA from T7-infected E. coli releases a DNA fragment consisting of the two termini of mature T7 DNA joined (this will be referred to as the termini-joined, or TJ, fragment) with one copy of the terminally repetitious sequence of T7 DNA (20, 21) in each TJ fragment (11). If the DNA sedimenting as concatemers in Figs. lb, c is concatemeric, the TJ fragment should be present in restriction enzyme digests of this DNA, and the ratio of the amount of
DNA in the TJ fragment to the amount of DNA in fragments from the termini of mature T7 DNA should increase with the distance sedimented by the DNA. To test this prediction, DNA in fractions collected from a preparative sedimentation performed as in Fig. lc was digested with MM; the resulting fragments were labeled with 32P and separated by agarose gel electrophoresis with detection by autoradiography. A digest of monomeric DNA (Fig. 2n) had fragments from the termini of mature T7 DNA, B and C fragments (Mb01 fragments of T7 DNA are mapped in (22), but the intensity of the band formed by the TJ fragment was comparatively weak in the digest of monomeric DNA. However, as the sedimentation rate of the DNA increased, the ratio of the amount of 32P in the TJ fragment to the amounts in the B and C fragments increased (Fig. 2, m-e), indicating that either concatemers, circles, or both are present in the DNA sedimenting more rapidly than monomeric DNA in the ethidium bromide-sucrose gradient. Because circular, monomeric T7 DNA is expected to sediment only 1.13 times as far as linear, monomeric T7 DNA (19), circles (if present) would have to be concatemeric or branched to sediment as far as the DNA in Fig. 2k-c. That at least some of the DNA digested for Fig. 2k-c is noncircular is indicated by the presence of bands formed by the terminal fragments of mature T7 DNA (B and C) in these fractions; data from electron microscopy presented below are in agreement. The length of the most rapidly sedimenting T7 concatemers in Figs. 1 and 2 is at least five times the length of T7 DNA. The results of Fig. 2 confirm the above conclusion that there is a substantial amount of T7 DNA in the regions between bands in Fig. 1, i.e., that the length of most concatemers varies. To further characterize the DNA sedimenting as T7 concatemers in Fig. 1, it was observed by electron microscopy. As shown in Table 1, most of the DNA is linear and unbranched, although a few linear, branched molecules were observed. No circular molecules of any kind were observed.
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FIG. 2. Mb01 digestion of concatemer-sedimenting fragments of 100 S+ DNA. The sample of 100 S+ DNA used in Fig. lc was sedimented through an ethidium bromide-sucrose gradient and fractionated by slow pipeting of successive layers from the top with a pipet 2 mm in inner diameter. A 15-pl portion of each undialyzed fraction was digested with restriction endonuclease Mb01 by adding an equal volume of a buffer containing 0.014 M Tris-Cl, pH 7.4, 0.016 M MgCl,, 100 pg/ml bovine serum albumen, 0.10% /3-mercaptoethanol, and enzyme (from New England Biolabs) diluted to a final concentration of 2 units/ml. Digestion was at 30’ for 2 hr. For detection of the resultant fragments, the ends of the digested DNA were labeled with 32P-labeled dCTP using the Klenow fragment of E. coli DNA polymerase I (25). Of the labeled digests, 2/3’s was subjected to agarose gel electrophoresis in a 0.7% ME agarose gel (Marine Colloids) at 0.45 V/cm for 16 hr, using procedures previously described (16). The gel was dried under vacuum and autoradiographed at -70’ with an image-intensifying screen (DuPont, Cronex). The fractions from which DNA was taken are indicated by letters a-p; the positions of monomeric T7 DNA, dimeric concatemers, and tetrameric concatemers are indicated at the top by 1, 2, and 4, respectively. Sample q is mature T7 DNA (2.5 ng), digested and labeled with 32P as described above. The arrowhead indicates the origin of electrophoresis; the vertical arrow the direction of electrophoresis; the horizontal arrow the direction of sedimentation.
This confirms the presence of T7 concatemers in the concatemer-sedimenting regions of the ethidium bromide-sucrose gradient of Fig. 1. These data provide the first evidence that at least some fragments of 100 S’ T7 DNA are concatemers. The finding of bands formed by a small amount of concatemer-sedimenting DNA with a uniform sedimentation rate in the presence of a background of DNA with a variable sedimentation rate was not reported in previous studies of 100 S DNA fragments. The structure of the region of 100 S+ DNA at which the nuclease Sl fragments were joined to 100 S+ DNA cannot be de-
duced from the data presented here. However, because of the evidence indicating that 100 S+ DNA is not topologically linear (this includes the observation that some regions of 100 S DNA are replicating) (14), forks in the structure must exist. Some double-stranded DNA segments in 100 S+ DNA, including the concatemers observed here, may be held together at these forks by single-stranded regions of DNA; other single-stranded regions may connect duplex DNA segments end-toend, as previously described (7, IO). Recombination and DNA repair may, together with DNA replication, produce some single-stranded regions in 100 S+ DNA
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1
ELECTRONMICROSCOPY OFCONCATEMER-SEDIMENTING, NUCLEASESl-RELEASED FRAGMENTSOF 100 S+ T7 DNA
Sample” n m i e
Linear, unbranched*
Linear, branched *
Circular*
Tangled”
32 26 36 26
2 2 0 0
0 0 0 0
25 26 32
10
’ Fraction in Fig. 2 from which DNA was taken for electron microscopy (13). Less than 25% of the DNA is host DNA (14). *Number of molecules. “Number of molecules too tangled to categorize.
(23). All of these processes can create single-stranded regions at random in the DNA and, therefore, can explain the appearance of concatemers with variable length in nuclease Sl digests of 100 S+ DNA. In theory, there are at least two possible mechanisms for the production in 100 S+ DNA of duplex DNA segments with lengths that are approximately integral multiples of the length of monomeric T7 DNA, such as the dimeric and tetrameric concatemers observed here: (1) end-to-end joining of linear products of T7 DNA replication by base pairing at single-stranded, repetitious termini, possibly with some regions at the termini left unpaired (7, 10); (2) replication of T7 DNA on a circular template (rolling circle model) and termination of replication after the template has been replicated one or three times (24). The absence of trimeric concatemers is difficult to explain if concatemers are produced by replication on a rolling circle. This observation can best be explained by end-to-end joining if the assumption is made that only the two progeny of a single replication event can terminally join. Thus, the data presented here favor possibility (I).
nical assistance, we thank Elena T. Moreno. For secretarial assistance, we thank Joanne Williams and Donna Scoggins. For a generous gift of unglucosylated T4 DNA, we thank Dr. J. W. Wiberg. For advice concerning the radiolabeling of T7 DNA, we thank Dr. Donna L. Montgomery. Support was received from the National Institutes of Health (Grant GM-24365) and from the Robert A. Welch Foundation (Grant AQ764).
REFERENCES I. THOMAS, C. A., KELLY, T. J., AND RHOADES,M., Cold Spring Harbor Symp. Quant. Biol. 33,
417-424 (1968). 2. CASJENS,S., AND KING, J., Annu. Rev. Biochem. 44, 555-611 (1975). 3. MURIALDO, H ., AND BECKER, A., Microbial. Rev. 42, 529-576 (1978). 4. EARNSHAW, W. C., AND CASJENS, S., Cell 21,
319-331 (1980). 5. KRUGER, D. H., AND SCHROEDER,C., Microbid. Rev. 46, 9-51, (1981). 6. KELLY, T. J., AND THOMAS, C. A., J. Mol. Biol. 44, 459-475 (1969). 7. SCHLEGEL,R. A., AND THOMAS, C. A., J. Mol. Biol. 68, 319-345 (1972). 8. DRESSLER,D., WOLFSON,J., AND MA&UN, M., Proc. Nat. Acad. Sci. USA 69,9981002 (1972). 9. WOLFSON,J. D., DRESSLER,D., AND MAGAZIN, M., Proc. Nat. Acad. Sci. USA 69, 499-504
(1971). WATSON, J. D., Nature New Biol. 239, 197-201 (1972). 11 LANGMAN,L., PAEXAU, V., SCRABA,D., MILLER, R. C., JR., ROEDER, G. S., AND SADOWSKI, P. D., Can&. J. Biochem. 66, 508-516 (1978). 12 SERWER,P., Virology 69, 89-107 (1974).
10.
ACKNOWLEDGMENTS
For assistance during the earlier stages of this work, we thank Nancy L. Smith. For assistance with electron microscopy we thank Shirly J. Hayes. For tech-
SHORT COMMUNICATIONS 13. SERWER, P., AND WATSON, R. H., Virology 108, 164-176 (1981). 14. SERWER, P., Virology 69, 70-88 (1974). 15. PAETKAU, V., LANGMAN, L., BRADLEY, R., SCRABA, D., AND MILLER, R. C., JR., J. Virol. 22.130-141 (1977). 16. SERWER, P., Biochemistry 19,3001-3004 (1980). 17. SERWER, P., AND GREENHAW, G. A., In “Electrophoresis ‘81” (R. C. Allen and P. Arnaud, eds.), pp. 627-633. DeGruyter, Berlin, 1981. 18. SERWER, P., GRAEF, P. R., AND GARRISON, P. N., Biochemistry 17,1166-1170 (1978). 19. HERSHEY, A. D., BURGI, E., AND INGRAHAM, L., Proc. Nat. Acad. Sci. USA 49, 748-755 (1963). 20. RITCHIE, D. A., THOMAS, C. A., JR., MACHATTIE,
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L. A., AND WENSINK, P. C., J. Mol. Biol. 23,
365-376 (1967). 21. DUNN, J. J., AND STUDIER, F. W., J. Mol. Biol. 148,303-330 (1981). 22. MCDONELL, M. W., SIMON, M. N., ANDSTUDIER, F. W., J. Mol. Biol. 110, 119-146 (1977). 23. BROKER, T., AND DOERMANN, A. H., Annu. Reu. Genet. 9,213-244 (1975). 24. GILBERT, W., AND DRESSLER, D., Cold Spring Harbor Symp. Quan. Biol. 33,473-484 (1968). 25. SMITH, M., LEUNG, D. W., GILLMAN, S., ASTELL, C. R., MONTGOMERY, D. L., AND HALL, B. D., Cell 16, 753-761(1979). 26. CLARK, R. W., WEVER, G. H. AND WIBERG, J. S. J. Virol. 33, 438-448.
123,4?4-479
Concatemers
(1982)
in a Rapidly Sedimenting,
Replicating
Bacteriophage
T7 DNA
PHILIP SERWER, GISELE A. GREENHAW, AND JERRY L. ALLEN Department
of Biochemistry,
The University
of Texas Health Science Center, San Antonio,
Texas 78284
Received August 2, 1982; accepted September 14, 1982 In previous studies, replicating bacteriophage T7 DNA was isolated as a rapidly sedimenting, multigenome-sized complex (100 S’ DNA), consisting of double-stranded DNA with some single-stranded regions. In the present study, 100 Sf DNA was digested with nuclease Sl and the products were fractionated by velocity sedimentation and analyzed by electron microscopy and digestion with restriction enzymes, followed by agarose gel electrophoresis. Some of the fragments released from 100 S+ DNA by nuclease Sl are linear, heterogeneous in length, and one to five times as long as mature T7 DNA. Uniformlength DNA’s one, two, and four times the length of mature T7 DNA were also present in sufficiently large amounts to form visible bands during velocity sedimentation.
During their assembly in uiuo, several bacteriophages with duplex DNA, including T4, T7, X, and P22, have been found to produce postreplicative molecules of DNA that are end-to-end aggregates of the mature bacteriophage DNA and that are referred to as concatemers (reviewed in (1-5). Concatemers produced by bacteriophage T7 have been fractionated by velocity sedimentation in sucrose gradients (6), and it was reported that these concatemers have single-stranded regions at the termini of some of their constituent mature genomes (7). Because of this observation and the observation that T7 DNA is linear during the first few rounds of replication (8, 9), joining of linear replication products by hydrogen bonding at single-stranded termini has been proposed to be the mechanism of formation of T7 concatemers (7, 10). However, subsequently a more random distribution of single-stranded regions in T7 concatemers has been proposed (12). If the latter study is correct, there is no direct evidence of terminal joining during the formation of T7 concatemers. Evidence has been presented indicating that T7 concatemers are packaged in a preformed T7 capsid and after packaging are cut to mature size (12, 13). Subsequent to the appearance of the linear replicative forms of T7 DNA described 0042-6822/82/160474-06$02.00/O Copyright All rights
0 19EZ by Academic Press, Inc. of reproduction in any form reserved.
474
in (8,9), replicating T7 DNA becomes part of a larger complex of DNA sedimenting at rates greater than 100 S and referred to as 100 S DNA (14); a more deproteinized form of 100 S+ DNA has been referred to as flowers (15). Digestion of 100 S DNA with nucleases specific for single-stranded DNA releases DNA sedimenting as, but not yet demonstrated to be, T7 concatemers (II, 14). As emphasized in (II), this DNA sediments as though it were heterogeneous in length, and, therefore, does not provide evidence of the terminal joining previously discussed (10). To further analyze fragments of 100 S+ T7 DNA, procedures of agarose gel electrophoresis capable of resolving T7 concatemers (16) were subsequently used to fractionate fragments of 100 S DNA released by nuclease Sl, specific for singlestranded DNA. It was found, in agreement with data previously obtained (11,14), that the mobility of most concatemer-migrating (and more slowly migrating) DNA released varied continuously. However, in contrast to the previous studies, it was found that a comparatively small amount of released DNA formed sharp bands at the positions of mature-length T7 DNA (monomeric DNA) and dimeric concatemers (17). If the concatemer-migrating DNA in (I 7) were really concatemeric,
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in the present study fragments of 100 S+ DNA sedimenting as concatemers have been characterized. In addition, the sedimentation profile of this DNA has been observed at a resolution higher than in previous studies. To fractionate and observe fragments of 100 S+ T7 DNA produced by digestion to completion with nuclease Sl, these fragments were sedimented in sucrose gradients containing 1 pg/ml ethidium bromide (ethidium bromide-sucrose gradients) and photographs of the long-wave ultraviolet light-induced fluorescence in the gradients were taken (18). As previously found using agarose gel electrophoresis (17), all regions of ethidium bromidesucrose gradients potentially containing concatemers isolated at 22 min after in-
these results would provide evidence of the joining by single DNA strands of monomeric DNA’s within concatemers, possibly the terminal joining previously discussed (10). The observation of concatemer-migrating DNA in (17) was accomplished by staining (without fractionating) gels, a procedure which facilitates the detection of faint bands; this procedure has not yet been used for the detection of fragments of 100 S DNA after sedimentation, although it is possible to do so (18). The concatemer-migrating fragments of 100 S+ DNA were not rigorously shown to be concatemeric in any of the above studies. Branched or circular DNA’s may migrate with concatemers during velocity sedimentation (19) and possibly also during agarose gel electrophoresis. Therefore,
menliscus -
-meniscus
-
-monomer -dimer -tetramer IT4
T7
T5 T4 -
a
b
c
FIG. 1. Sedimentation of nuclease Sl-fragmented 100 S+ T7 DNA. A loo-ml culture of E. coli BB/ 1 was infected with bacteriophage T7; 40-ml portions of this culture were chilled at 22 and 25 min after infection and 100 S+ DNA was prepared from both after lysis with Sarkosyl, as previously described (17). A 60-~1, undialyzed portion of both 100 S DNA preparations, containing DNA from 1.4 X 10’ cells, was digested with 100 units/ml nuclease Sl (Sigma) as previously described (17). After dialysis against 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 M EDTA for 16 hr and addition of 0.25 pg of bacteriophage T4 DNA (18), the digested DNA’s were sedimented through ethidium bromide-sucrose gradients in the above buffer, followed by photography of fluorescence, as previously described (18). Also sedimented was a sample containing 0.40 pg each of DNA’s from bacteriophages T4, T5, and 0.25 pg of DNA from bacteriophage T7 (18). (a) T4, T5, and T7 DNA, (b) 100 S+ DNA digest, 22 min; (c) 106 S+ DNA digest, 25 min. The dependence of distance sedimented in ethidium bromide-sucrose gradients on the M, of duplex DNA was previously established using the mature DNA’s of bacteriophages T4, T5, and T7 as standards (18). However, T4 DNA is glucosylated; T5 and T7 DNA’s are not. In (18), correction for the effect of glucosylation of T4 DNA was not made. When this is done, the exponent in Eq. 3 of Ref. (18) is changed to 0.36; this revised exponent was used for all calculations made here. Unglucosylated T4 DNA (26) sedimented 7 f 1% slower than glucosylated T4 DNA.
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fection (Fig. lb) and at 25 min after infection (Fig. lc) were more fluorescent than the background regions of a gradient containing a mixture of mature DNA’s from bacteriophages T4, T5, and T7 (Fig. la). This indicates that molecules too variable in sedimentation rate to form sharp bands are present in the gradients of Figs. lb, c. However, superimposed on this continuously varying background were three bands closer to the origin of sedimentation than T4 DNA added to the samples in Figs. lb, c as an internal standard for molecular weight (A4,) (by sedimentation in a separate gradient without the T4 DNA, it was shown that no band formed by fragments of 100 S+ DNA is at the position of the T4 DNA standard). Correcting the position of T4 DNA for glucosylation of this DNA (legend to Fig. l), the positions of the bands formed by fragments of 100 S+ DNA are calculated (legend to Fig. 1) to be the positions of linear, duplex DNA molecules with MZ’s that are 0.93 +- 0.1 (least rapidly sedimenting), 2.0 + 0.1, and 4.2 + 0.2 (most rapidly sedimenting) times the M, of mature T7 DNA. These ratios are not significantly different from 1, 2, and 4; evidence is presented below suggesting that DNA’s forming the bands are T7 concatemers. Therefore, DNA’s forming the two bands furthest from the origin of sedimentation will be referred to as dimeric concatemers and tetrameric concatemers, respectively; DNA forming the band closest to the origin of sedimentation will be referred to as monomeric DNA. A possible interpretation of the absence of a band formed by “trimeric concatemers” is discussed below. It has previously been shown that restriction enzyme digestion of unfractionated DNA from T7-infected E. coli releases a DNA fragment consisting of the two termini of mature T7 DNA joined (this will be referred to as the termini-joined, or TJ, fragment) with one copy of the terminally repetitious sequence of T7 DNA (20, 21) in each TJ fragment (11). If the DNA sedimenting as concatemers in Figs. lb, c is concatemeric, the TJ fragment should be present in restriction enzyme digests of this DNA, and the ratio of the amount of
DNA in the TJ fragment to the amount of DNA in fragments from the termini of mature T7 DNA should increase with the distance sedimented by the DNA. To test this prediction, DNA in fractions collected from a preparative sedimentation performed as in Fig. lc was digested with MM; the resulting fragments were labeled with 32P and separated by agarose gel electrophoresis with detection by autoradiography. A digest of monomeric DNA (Fig. 2n) had fragments from the termini of mature T7 DNA, B and C fragments (Mb01 fragments of T7 DNA are mapped in (22), but the intensity of the band formed by the TJ fragment was comparatively weak in the digest of monomeric DNA. However, as the sedimentation rate of the DNA increased, the ratio of the amount of 32P in the TJ fragment to the amounts in the B and C fragments increased (Fig. 2, m-e), indicating that either concatemers, circles, or both are present in the DNA sedimenting more rapidly than monomeric DNA in the ethidium bromide-sucrose gradient. Because circular, monomeric T7 DNA is expected to sediment only 1.13 times as far as linear, monomeric T7 DNA (19), circles (if present) would have to be concatemeric or branched to sediment as far as the DNA in Fig. 2k-c. That at least some of the DNA digested for Fig. 2k-c is noncircular is indicated by the presence of bands formed by the terminal fragments of mature T7 DNA (B and C) in these fractions; data from electron microscopy presented below are in agreement. The length of the most rapidly sedimenting T7 concatemers in Figs. 1 and 2 is at least five times the length of T7 DNA. The results of Fig. 2 confirm the above conclusion that there is a substantial amount of T7 DNA in the regions between bands in Fig. 1, i.e., that the length of most concatemers varies. To further characterize the DNA sedimenting as T7 concatemers in Fig. 1, it was observed by electron microscopy. As shown in Table 1, most of the DNA is linear and unbranched, although a few linear, branched molecules were observed. No circular molecules of any kind were observed.
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jklmnopq
FIG. 2. Mb01 digestion of concatemer-sedimenting fragments of 100 S+ DNA. The sample of 100 S+ DNA used in Fig. lc was sedimented through an ethidium bromide-sucrose gradient and fractionated by slow pipeting of successive layers from the top with a pipet 2 mm in inner diameter. A 15-pl portion of each undialyzed fraction was digested with restriction endonuclease Mb01 by adding an equal volume of a buffer containing 0.014 M Tris-Cl, pH 7.4, 0.016 M MgCl,, 100 pg/ml bovine serum albumen, 0.10% /3-mercaptoethanol, and enzyme (from New England Biolabs) diluted to a final concentration of 2 units/ml. Digestion was at 30’ for 2 hr. For detection of the resultant fragments, the ends of the digested DNA were labeled with 32P-labeled dCTP using the Klenow fragment of E. coli DNA polymerase I (25). Of the labeled digests, 2/3’s was subjected to agarose gel electrophoresis in a 0.7% ME agarose gel (Marine Colloids) at 0.45 V/cm for 16 hr, using procedures previously described (16). The gel was dried under vacuum and autoradiographed at -70’ with an image-intensifying screen (DuPont, Cronex). The fractions from which DNA was taken are indicated by letters a-p; the positions of monomeric T7 DNA, dimeric concatemers, and tetrameric concatemers are indicated at the top by 1, 2, and 4, respectively. Sample q is mature T7 DNA (2.5 ng), digested and labeled with 32P as described above. The arrowhead indicates the origin of electrophoresis; the vertical arrow the direction of electrophoresis; the horizontal arrow the direction of sedimentation.
This confirms the presence of T7 concatemers in the concatemer-sedimenting regions of the ethidium bromide-sucrose gradient of Fig. 1. These data provide the first evidence that at least some fragments of 100 S’ T7 DNA are concatemers. The finding of bands formed by a small amount of concatemer-sedimenting DNA with a uniform sedimentation rate in the presence of a background of DNA with a variable sedimentation rate was not reported in previous studies of 100 S DNA fragments. The structure of the region of 100 S+ DNA at which the nuclease Sl fragments were joined to 100 S+ DNA cannot be de-
duced from the data presented here. However, because of the evidence indicating that 100 S+ DNA is not topologically linear (this includes the observation that some regions of 100 S DNA are replicating) (14), forks in the structure must exist. Some double-stranded DNA segments in 100 S+ DNA, including the concatemers observed here, may be held together at these forks by single-stranded regions of DNA; other single-stranded regions may connect duplex DNA segments end-toend, as previously described (7, IO). Recombination and DNA repair may, together with DNA replication, produce some single-stranded regions in 100 S+ DNA
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COMMUNICATIONS TABLE
1
ELECTRONMICROSCOPY OFCONCATEMER-SEDIMENTING, NUCLEASESl-RELEASED FRAGMENTSOF 100 S+ T7 DNA
Sample” n m i e
Linear, unbranched*
Linear, branched *
Circular*
Tangled”
32 26 36 26
2 2 0 0
0 0 0 0
25 26 32
10
’ Fraction in Fig. 2 from which DNA was taken for electron microscopy (13). Less than 25% of the DNA is host DNA (14). *Number of molecules. “Number of molecules too tangled to categorize.
(23). All of these processes can create single-stranded regions at random in the DNA and, therefore, can explain the appearance of concatemers with variable length in nuclease Sl digests of 100 S+ DNA. In theory, there are at least two possible mechanisms for the production in 100 S+ DNA of duplex DNA segments with lengths that are approximately integral multiples of the length of monomeric T7 DNA, such as the dimeric and tetrameric concatemers observed here: (1) end-to-end joining of linear products of T7 DNA replication by base pairing at single-stranded, repetitious termini, possibly with some regions at the termini left unpaired (7, 10); (2) replication of T7 DNA on a circular template (rolling circle model) and termination of replication after the template has been replicated one or three times (24). The absence of trimeric concatemers is difficult to explain if concatemers are produced by replication on a rolling circle. This observation can best be explained by end-to-end joining if the assumption is made that only the two progeny of a single replication event can terminally join. Thus, the data presented here favor possibility (I).
nical assistance, we thank Elena T. Moreno. For secretarial assistance, we thank Joanne Williams and Donna Scoggins. For a generous gift of unglucosylated T4 DNA, we thank Dr. J. W. Wiberg. For advice concerning the radiolabeling of T7 DNA, we thank Dr. Donna L. Montgomery. Support was received from the National Institutes of Health (Grant GM-24365) and from the Robert A. Welch Foundation (Grant AQ764).
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10.
ACKNOWLEDGMENTS
For assistance during the earlier stages of this work, we thank Nancy L. Smith. For assistance with electron microscopy we thank Shirly J. Hayes. For tech-
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