8
Cell, Vol. 21, 277-284. August 1980. Copyright 0 1980 by MIT Tandem Repeats within the Inverted Terminal Repetition of Vaccinia Virus DNA Riccardo Wittek and Bernard Moss Laboratory of Biology of Viruses National Institute of Allergy and Infectious Diseases Bethesda, Maryland 20205 Summary A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approxi- mately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restric- tion analysis of cloned DNA fragments and by hy- bridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abun- dance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites oc- curred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of ap- proximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence con- taining single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replica- tion. Introduction Poxviruses comprise a large group of DNA viruses that are distinguished by their great size, cytoplasmic site of replication and possession of a virion-associ- ated transcriptase (see review by Moss, 1974). Little information is currently available regarding the orga- nization or mode of replication of the poxvirus ge- nome. Vaccinia virus, the most intensively studied member of this group, contains a nonpermuted linear double-stranded DNA molecule of approximately 120 million daltons (Geshelin and Berns, 1974; Wittek et al., 1977). In addition to its large size, vaccinia virus DNA has some unusual features. First, the comple- mentary strands of DNA are terminally cross-linked, so that a single-stranded circle is formed upon dena- turation (Geshelin and Berns, 1974). Second, a stretch of about 10,000 nucleotides at one end of each strand of vaccinia virus DNA is complementary to the nucleotide sequence at the other end, thus forming an inverted terminal repetition (Garon, Bar- bosa and Moss, 1978; Wittek et al., 1978a). The possibility of additional repetitive elements within the vaccinia virus genome was suggested by the rate of reannealing of sheared DNA fragments (Grady and Paoletti, 1977; Pedrali-Noy and Weissbach, 1977). In this paper, we demonstrate blocks of tandemly re- peated sequences within the inverted terminal repeti- tion and suggest that they function to accelerate cy- clization of single strand8 of DNA during replication. Results Detection of a Repeated Sequence within Vaccinia Virus DNA We searched for a repeated sequence within the vaccinia virus genome by cleaving the DNA with a variety of restriction endonucleases and analyzing the fragments by polyacrylamide gel electrophoresis. As shown in Figure 1, an intense ethidium bromide-stain- ing band was detected when Hinf I, Taq I and Mbo II were used. The size of the fragment in each case was estimated to be 70 bp. Generation of an identical size fragment with several restriction endonucleases sug- gested that the same sequence was repeated in a tandem array. Localization of the Repeats within the Inverted Terminal Repetition We suspected that the repeats might form part of the inverted terminal repetition, which in the WR strain of vaccinia virus is approximately 10,300 bp in length (Garon et al., 1978). Evaluation of this possibility was facilitated by our recent cloning in bacteriophage lambda of a 9000 bp terminal Eco RI fragment from vaccinia virus DNA (Wittek et al., 1980a). Hinf I digests of the recombinant DNA as well as the purified vacci- nia virus DNA insert showed clearly that the 70 bp reiterated sequence is present within the inverted terminal repetition (Fig. 2). Our next experiment served both to demonstrate that the repeat sequence is present exclusively in the inverted terminal repetition and to localize it further within that segment of DNA. The 70 bp fragment formed by Hinf I digestion of the recombinant DNA was isolated by polyacrylamide gel electrophoresis and labeled by filling in the staggered ends with DNA polymerase and s-32P-labeled deoxyribonucleoside triphosphates. The fragment was then used as a ra- dioactive probe to detect the repeated sequence in separated fragments of vaccinia virus DNA that had been immobilized on nitrocellulose by a blotting pro- cedure (Southern, 1975). Results obtained with Hind Ill and Sal I digests of vaccinia virus DNA are shown in Figure 3. Hind Ill digestion of vaccinia virus DNA results in the formation of 15 fragments. Of these, only the second and third largest, which are derived from the two ends of the genome (Garon et al., 19781, were labeled with the probe. Sal I digestion of vaccinia

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

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Cell, Vol. 21, 277-284. August 1980. Copyright 0 1980 by MIT

Tandem Repeats within the Inverted Terminal Repetition of Vaccinia Virus DNA

Riccardo Wittek and Bernard Moss Laboratory of Biology of Viruses National Institute of Allergy and Infectious Diseases Bethesda, Maryland 20205

Summary

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approxi- mately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restric- tion analysis of cloned DNA fragments and by hy- bridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abun- dance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites oc- curred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of ap- proximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence con- taining single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replica- tion.

Introduction

Poxviruses comprise a large group of DNA viruses that are distinguished by their great size, cytoplasmic site of replication and possession of a virion-associ- ated transcriptase (see review by Moss, 1974). Little information is currently available regarding the orga- nization or mode of replication of the poxvirus ge- nome. Vaccinia virus, the most intensively studied member of this group, contains a nonpermuted linear double-stranded DNA molecule of approximately 120 million daltons (Geshelin and Berns, 1974; Wittek et al., 1977). In addition to its large size, vaccinia virus DNA has some unusual features. First, the comple- mentary strands of DNA are terminally cross-linked, so that a single-stranded circle is formed upon dena- turation (Geshelin and Berns, 1974). Second, a stretch of about 10,000 nucleotides at one end of each strand of vaccinia virus DNA is complementary to the nucleotide sequence at the other end, thus forming an inverted terminal repetition (Garon, Bar- bosa and Moss, 1978; Wittek et al., 1978a). The possibility of additional repetitive elements within the

vaccinia virus genome was suggested by the rate of reannealing of sheared DNA fragments (Grady and Paoletti, 1977; Pedrali-Noy and Weissbach, 1977). In this paper, we demonstrate blocks of tandemly re- peated sequences within the inverted terminal repeti- tion and suggest that they function to accelerate cy- clization of single strand8 of DNA during replication.

Results

Detection of a Repeated Sequence within Vaccinia Virus DNA We searched for a repeated sequence within the vaccinia virus genome by cleaving the DNA with a variety of restriction endonucleases and analyzing the fragments by polyacrylamide gel electrophoresis. As shown in Figure 1, an intense ethidium bromide-stain- ing band was detected when Hinf I, Taq I and Mbo II were used. The size of the fragment in each case was estimated to be 70 bp. Generation of an identical size fragment with several restriction endonucleases sug- gested that the same sequence was repeated in a tandem array.

Localization of the Repeats within the Inverted Terminal Repetition We suspected that the repeats might form part of the inverted terminal repetition, which in the WR strain of vaccinia virus is approximately 10,300 bp in length (Garon et al., 1978). Evaluation of this possibility was facilitated by our recent cloning in bacteriophage lambda of a 9000 bp terminal Eco RI fragment from vaccinia virus DNA (Wittek et al., 1980a). Hinf I digests of the recombinant DNA as well as the purified vacci- nia virus DNA insert showed clearly that the 70 bp reiterated sequence is present within the inverted terminal repetition (Fig. 2).

Our next experiment served both to demonstrate that the repeat sequence is present exclusively in the inverted terminal repetition and to localize it further within that segment of DNA. The 70 bp fragment formed by Hinf I digestion of the recombinant DNA was isolated by polyacrylamide gel electrophoresis and labeled by filling in the staggered ends with DNA polymerase and s-32P-labeled deoxyribonucleoside triphosphates. The fragment was then used as a ra- dioactive probe to detect the repeated sequence in separated fragments of vaccinia virus DNA that had been immobilized on nitrocellulose by a blotting pro- cedure (Southern, 1975). Results obtained with Hind Ill and Sal I digests of vaccinia virus DNA are shown in Figure 3. Hind Ill digestion of vaccinia virus DNA results in the formation of 15 fragments. Of these, only the second and third largest, which are derived from the two ends of the genome (Garon et al., 19781, were labeled with the probe. Sal I digestion of vaccinia

Cell 278

Alu I Hae 111 Hinf I Hpa II Mbo I Mbo II Taa I Figure 1. Restriction Endonuclease Digests of Vaccinia Virus DNA

The indicated restriction endonucleases were used to digest 2 pg samples of vaccinia virus DNA. After electrophoresis in 10% polyacryl- amide gels, DNA fragments were visualized by staining with ethidium bromide. The track at the extreme right contains a Hae Ill digest of pER322 DNA; the lengths of fragments are indicated in bp.

-104

-89 -80

-64 -57

virus DNA also results in the generation of a large number of fragments, of which a 3600 bp fragment is present in double molar amount and is derived from the extreme end of the terminal repetition (Garon et al., 1978). Significantly, only this fragment was la- beled with the 70 bp probe (Figure 3). Thus the repeats were localized within the outermost 3600 bp of the inverted terminal repetition.

End Labeling of the Repeats Since ethidium bromide staining is difficult to quanti- tate, an alternative procedure was used to estimate the number of copies of the repeat sequence. Hinf I and Taq I digests of vaccinia virus DNA and of the purified cloned terminal fragment were end-labeled by the polynucleotide kinase exchange procedure, which previously was shown to label such fragments equally (Berkner and Folk, 1977). An autoradiograph of end- labeled fragments (Figure 4) demonstrated the high degree of reiteration of the 70 bp fragment much more dramatically than our previous ethidium bromide- stained gels. Densitometry of the autoradiograph of the end-labeled Hinf I digest of $X1 74 DNA confirmed that the exchange reaction labeled all the termini equally. Quantitation of densitometer tracings of the end-labeled Hinf I digests of total vaccinia virus DNA and of the cloned terminal fragment was rendered difficult, however, by the extreme difference in mag- nitude of the peaks corresponding to the repetitive and presumed unique sequences. Nevertheless, we did estimate that the 70 bp fragment was present at least 30 times per cloned terminal repetition.

Arrangement of Repeated Sequences within the Inverted Terminal Repetition The experiments described so far have enabled us to localize and determine the approximate number of copies of the repeated sequence within the 10,300 bp

of the inverted terminal repetition of vaccinia virus DNA. To obtain more precise information, the Hinf I sites within the inverted terminal repetition were mapped by a partial digestion procedure (Smith and Birnstiel, 1976). The cloned Eco RI fragment was labeled at both ends using DNA polymerase and (Y- 32P-deoxyribonucleoside triphosphates. It was then cleaved at the single Xho I site and the two fragments (Xho I-A and Xho I-B) were isolated. The larger one (Xho I-A) contains the sequences nearer to the end of the vaccinia virus genome. Both fragments were then digested for several different intervals of time using limiting amounts of Hinf I. The partial digestion prod- ucts were separated by agarose gel electrophoresis and those containing the labeled end were detected by autoradiography. In Figure 5, the tracks labeled 0 show undigested Xho I-A or Xho I-B fragments, the tracks labeled 5’, 20’ and 4O’show their partial diges- tion products and the tracks labeled C show their complete digestion products. Tandem repetitions are seen as arrays of regularly spaced bands in the partial digests. There are two such arrays within the partial digestion products of the terminal Xho I-A fragment (Figure 5A). The lower one contains 13 regularly spaced bands and the upper one contains 17. No further regularity in the spacing of bands occurs above that point, nor is there any evidence of repetitions within the Xho I-B fragment (Fig 5B). This result is consistent with the localization of repetitions within the terminal Sal I fragment by blot hybridization (Fig- ure 3). The first Hinf I site in the Xho I-A fragment was located approximately 150 bp from the end of the DNA and the last Hinf I site in the first set of repeats was approximately 1000 bp from the end. From this difference, we calculated that a spacing of 70 bp occurred between successive Hinf I sites. That num- ber is in agreement with the 70 bp size determined for the multimolar fragment produced upon complete

Tandem Repeats in Vaccinia Virus DNA 279

1 2 3 Figure 2. Hinf I Digest of (1) Vaccinia Virus DNA, (2) Recombinant DNA Containing 9000 bp Eco RI End Fragment of Vaccinia Virus DNA and (3) Vaccinia Virus DNA Insert Purified from above Recombinant

Ethidium bromide-stained 10% polyacqlamide gel is shown.

cleavage of vaccinia virus DNA with Hinf I, Taq l or Mbo II. The same spacing was also found between the Hinf I sites within the second set of 17 bands. Partial Taq I and Mbo II digestion also produced two sets of repeating sequences with the same spacing, strongly suggesting that the repeated sequence in both blocks is very similar if not identical. Thus there are a total of 30 (13 + 17) repetitions at each end of the genome. This value is similar but more reliable than the value obtained by integration of the peaks from Figure 4.

To determine whether the 435 bp segment of DNA separating the two blocks of repeats contains a unique sequence or one with different repeating units, we screened a large number of restriction endonucleases for their ability to cleave the end-labeled Xho I-A fragment. Three enzymes, Dde I, Alu I and Sau 3A,

1 2 Hind Ill

1 2 Sal I

Figure 3. Autoradiograph of Hind Ill and Sal I Digests of Vaccinia Virus DNA Immobilized on Nitrocetlulose Membrane

The filters were probed with 32P-labeled (1) total vaccinia virus DNA and (2) 70 bp Hinf I fragment.

were found to cleave at single sites within the 435 bp sequence. The result obtained with Dde I is shown in Figure 6. The size of the labeled terminal fragment obtained by complete digestion was about 1100 bp, indicating that the first Dde I site occurred just beyond the first set of tandem repeats. The next Dde I site occurred about 3600 bp from the end of the DNA which is beyond the second set of repeats. We there- fore concluded that the region between the two blocks of repeats is composed of a unique DNA sequence. A map of Hinf I, Alu I, Dde I and Sal I sites within the terminal region of vaccinia DNA is shown in Figure 7.

Comparison of the Size of End Fragments from Cloned DNA and Virion DNA Since cloning of the terminal fragment involved single- strand DNAase digestion to remove the natural cross- link (Wittek et al., 1960a), it was important to deter-

Cell 200

9.46 - 6.67 -

4.26 - 231

141

07

54

33

1 2 ox 1 2@X

Hinf I Taq I Figure 4. Autoradiograph of End-Labeled Hinf I and Taq I Fragments

Separate Hinf I and Taq I digests of (1) vaccinia virus DNA, (2) cloned 9000 bp Eco RI end fragment from vaccinia virus DNA and (3) +X174RF DNA were end-labeled by the polynucleotide kinase ex- change procedure. DNA fragments were resolved by electrophoresis on a 10% polyacrylamide gel. An autoradiograph is shown. The sizes in bp of the @Xl 74 DNA Taq I fragments are listed, and the sizes of the resotved @Xi 74 Hinf I fragments are 249, 200, 151, 140. 116, 100. 82. 66 (double molar fragment). 48, 42 and 40 (Sanger et al., 1977).

mine whether significant amounts of DNA were lost. To estimate this, the 3600 bp Sal I end fragment from vaccinia virus DNA and the corresponding piece of cloned DNA were purified by agarose gel electropho- resis. The two were then cleaved with Dde I and Alu I, both of which cut within the 435 bp sequence separating the two sets of repeats (Figure 7). The digestion products were compared by electrophoresis on the same agarose gel along with appropriate size markers. As shown in Figure 8, the uncleaved Sal I end fragments of cloned and virion DNA have almost the same mobility. Cleavage of the Sal I end fragments with Alu I resulted in the formation of two fragments in each case. The larger ones co-migrated and had a size estimated to be 2260 bp. In contrast, the smaller ones, which are derived from the end of the genome, have slightly different mobilities. The Alu I end frag- ment from cloned DNA was estimated to be 1340 bp while the analogous fragment from virion DNA was 1390 bp.

::1- - 1.353 - 1.078

- 372

- .6w

[: 1

f$ z *

I ;#j

0 Brn4o’C A@XC Wzo,B 0

A B

Figure 5. Autoradiograph of Partial Hinf I Digests

The recombinant DNA was cleaved with Eco RI and all fragments were end-labeled using a-32P-deoxyribonucleoside triphosphates and DNA polymerase. The fragments were then cleaved with Xho I and separated on an agarose gel. The two Xho I fragments (A. 6) derived from the vaccinia DNA insert were purified and partially digested with Hinf I followed by electrophoresis on a 1.2% agarose gel. The sizes (kb) of end-labeled X-Hind Ill and +X174-Hae Ill restriction fragments used as molecular weight markers are shown on the left and right, respectively. (0) Portion of the incubation mixture before the addition of enzyme; (C) complete digestion with excess enzyme.

9.46 -

6.67 -

4.26 -

2.25- 1.96 -

- 1.353

- 1.078

A 0 5’ 2tY 4(y c QX

Figure 6. Autoradiograph of Partial Dde I Digests

The end-labeled Xho I-A fragment was purified as described in the legend to Figure 5. The fragment was then partially cleaved with Dde I and the products were separated by electrophoresis on a 1.2% agarose gel. Symbols are as in the legend to Figure 5.

Two fragments were also obtained upon cleavage of the Sal I end fragments with Dde I. The larger fragments of both DNAs co-migrated and their size was estimated to be 2480 bp. Again, the smaller end

Tandem Repeats in Vaccinia Virus DNA 281

I I I I I I I I I II I I III,lI,,IIl,II,,I I A b

1 Figure 7. Location of Hinf I, Alu I, Dde I and Sal I Sites within the Terminal Region of Vac-

I I 1 I I I I cinia Virus DNA 0

5240,

4%L

3380 -

m> 1620 - 1330 -

1:5 210 KILOBASE PAIRS

2.5

-1353

- 1078

- 872

- 603

AABABABQX

Alu I Dde I Figure 8. Restriction Endonuclease Digests of Sal I End Fragments Isolated from (A) Vaccinia Virus DNA or (6) the Cloned Fragment

The end fragment of vaccinia virus DNA was obtained from a complete Sal l digest. The Sal I end fragment of cloned DNA was obtained by cleaving the recombinant DNA with Eco RI. Sal I and Xho I. (Cleavage with Eco RI was necessary to separate the cloned DNA from the X arms; cleavage with Xho I was needed to remove a second Sal I fragment of vaccinia DNA co-migrating with the end fragment.) After digestion of the purified Sal I fragments with the enzymes indicated, the fragments were separated on a 1% agarose gel and visualized by staining with ethidium bromide. The sizes (bp) of A-Hind III/Eco RI and @Xl 74-Hae Ill restriction fragments are shown on the left and right. respectively.

fragments appeared to differ in size. The Dde I end fragment from cloned DNA was 1090 bp whereas the analogous fragment from virion DNA was 1140 bp.

Thus the difference in size between authentic and cloned vaccinia virus DNA appears to be approxi- mately 50 bp and is due to a loss of sequences within the 1140 bp nearest the end of the DNA. The amount of DNA lost is less than one repeat unit and is probably due to removal of terminal nucleotides during DNAase treatment prior to cloning. The demonstration of sharp discrete bands upon Dde I and Alu I digestion of DNA from the recombinant phage and from vaccinia virus also serves to demonstrate the fidelity with which the repeating units are replicated.

Discussion

The studies described here demonstrate that a se- quence of approximately 70 bp containing Hinf I, Taq I and Mbo II restriction endonuclease sites is repeated 60 times within the vaccinia virus genome. This feature is not peculiar to the WR strain of vaccinia virus since recent experiments carried out in our laboratory indi-

3:o 3.5 Symbols: (1) Hinf I; (&) Dde I; (6) Alu I; (W Sal I.

cate that a similar or identical length sequence, with the same three restriction sites, is tandemly repeated in the DNA of the Lister strain of vaccinia virus and rabbitpox virus. Hybridization studies, using the puri- fied 70 bp fragment labeled with 32P as a probe, served to localize this sequence within the two end fragments of vaccinia virus DNA and further demon- strated that it was located near the outer end of the 10,300 bp inverted terminal repetition. Thus, of the total 182,000 bp in the vaccinia virus genome, 4200 consist of a 70 bp sequence repeated 60 times and 16,400 consist of an 8200 bp sequence (that is, the remainder of the inverted terminal repetition) repeated two times. This complex organization probably ac- counts for previous conclusions, based solely on reas- sociation kinetics, that 7640 bp of the genome consist of a 7 fold repeat (Grady and Paoletti, 1977) or that 12,740 bp consist of a 10 fold repeat (Pedrali-Noy and Weissbach, 1977).

Precise mapping of the tandem repeats was facili- tated by using DNA cloned in bacteriophage lambda. We demonstrated that the Hinf I repeats occur in two blocks of 13 and 17 tandemly arranged units sepa- rated by an intervening sequence of approximately 435 bp containing unique Dde I, Alu I and Sau 3A sites. Except for minor differences, the patterns ob- tained when end-labeled DNA was digested with Taq I and Mbo II were remarkably similar to that obtained with Hinf I (our unpublished data). Two additional bands, one at the end of the first set of repeats and one at the start of the second, were found with Taq I. Three bands within the first set of repeats appeared to be missing when Mbo II was used although the spacing of all of the others appeared identical. These differences could result from single base substitu- tions. We believe that the 70 bp sequence occurs in the same orientation in both sets of repeats, since self-annea,ling of single-stranded end fragments of vaccinia virus DNA was not seen (Garon et al., 1978).

The first repetitive Hinf I site occurred approximately 150 bp from the end of the cloned DNA. However, the distance of this first Hinf I site to the end of the genome might be slightly greater since terminal Dde I and Alu I fragments from virion DNA migrated slightly slower than analogous fragments from cloned DNA during agarose gel electrophoresis. This difference was at- tributed to removal of approximately 50 bp from the end of the genome during single-strand DNAase treat- ment, which was necessary to remove the terminal cross-linked portion of the DNA prior to cloning. It is possible, however, that the difference in length is actually less than 50 bp and that DNA fragments

Cell 282

containing the cross-link have an anomalous migration rate.

Further comparisons between cloned and virion DNA indicate little or no difference in the number or arrangement of tandem repetitions. Mapping of the repetitions in virion DNA is made difficult, however, by the inability to label the cross-linked end. Indeed, the existence of tandem repetitions was originally missed because the label was introduced too far from the end of the DNA to resolve them (Wittek et al., 1978a). In an experiment performed in this laboratory by J. Cooper (personal communication), the terminal 3600 bp Sal I fragment from virion DNA, which still con- tained the terminal cross-link, was labeled at the site of restriction endonuclease cleavage, and partial Hinf I digestion products were analyzed by agarose gel electrophoresis. Two sets of repeats separated by an intervening sequence were clearly seen. However, because the site of labeling was 3600 bp from the terminus of the genome, individual bands could be counted accurately only in the 17 unit repetition. Nevertheless, the difference in size between the ter- minal Dde I fragments of cloned and virion DNA is less than one repeating unit, indicating that the other block of repeats contains the same 13 units as cloned DNA.

Since the virus used to extract DNA for restriction endonuclease analysis has been passaged several times after plaque purification, the number and ar- rangement of repeats seem to be fairly stable. How- ever, given the potential for recombination, we would not be surprised if other plaque isolates did not have exactly the same numbers of repeats. In this regard, we have found isolates with slightly different length terminal Dde I and Alu I fragment8 (our unpublished results). Whether this also can account for the length heterogeneity of longer terminal restriction fragments found in virus that had not been plaque-purified (Wit- tek et al., 1978b) or had been passaged repeatedly (McCarron et al., 1978) remains to be determined.

A special mechanism must exist to maintain the 10,300 bp sequence complementarity at the two ends of the vaccinia virus genome and to account for the observation that a small deletion occurring near one end of the genome is reflected as a mirrow image deletion at the other end (McFadden and Dales, 1979). In this regard, Sambrook [cited by Daniel1 (197611 and Lechner and Kelly (1977) have suggested models for replication of adenovirus DNA, which may also be applicable to vaccinia virus. According to this model, initiation at the 3’ end of a displaced adenovi- rus DNA strand can proceed via a circular intermedi- ate formed by hybridization of its 100 bp inverted terminal repetition. Indeed, the terminal redundancy of adenovirus was first recognized by its ability to form such structures in vitro (Wolfson and Dressler, 1972; Garon, Berry and Rose, 1972). Similar single- stranded circles can also be formed in vitro with vaccinia virus DNA, provided the cross-links at the ends are removed (Garon et al., 1978). Although there

is relatively little information available regarding the precise mechanism of vaccinia virus DNA replication, removal of both cross-links from the parental genome appears to be a very early step (Pago, 1977) and formation of cross-links a very late step in the matu- ration of progeny DNA (Esteban and Holowczak, 1977). Accordingly, a single-stranded circular DNA intermediate could be involved in vaccinia virus repli- cation.

Although the large size of the vaccinia virus ge- nome, compared to that of adenovirus, would tend to decrease the rate of cyclization of single strands of DNA by about 10 fold (Jacobson and Stockmayer, 1950; Wang and Davidson, 19661, the much longer complementary regions and particularly the presence of tandem repeats should enormously increase the rate of nucleation (Wetmur and Davidson, 1968). We propose a model (Figure 9) in which the nucleation event consists of the annealing of any 70 nucleotide repeat at one end with any repeat at the other end of

TANDEM REPEATS

UNIQUE TANDEM REPEATS

-- - -- -------m

. / . /

INVERTED

I

INVERTED REPEAT REPEAT

ANNEALING OF

t

TANDEM REPEATS

ANNEALING OF INVERTED REPEAT

Figure 9. Model Showing the Cyclization of Single Strands of Vac- cinia Virus DNA That Are Displaced during Replication

The nucleation event consists of the annealing of 70 bp repeats. This is followed by hybridization of the remaining 7000 nucleotide portion of the inverted terminal repetition and final realignment to maximize base pairing.

Tandem Repeats in Vaccinia Virus DNA 283

the strand. The next steps consist of the annealing of adjacent repeats and the remaining 7000 nucleotide portion of the inverted terminal repetition. We suggest that the latter holds the structure together while tan- dem repeats are realigned so as to maximize base pairing. This realignment step might be favored by the presence of short unique regions at the very end of the DNA and between the two blocks of tandem re- peats. That precise alignment does occur was dem- onstrated by electron microscopy and single-stranded nuclease digestion prior to our knowledge of the tan- dem repeats (Garon et al., 1978).

Preliminary studies suggest that the terminal region of the vaccinia virus genome may have another un- usual feature. When the end-labeled Xho I-A fragment of cloned DNA was digested to completion with Hinf I, a single-labeled 150 bp fragment was found. Un- expectedly, when Taq I was used, two smaller labeled bands were detected (not shown). Such a result could occur if the terminal region contains more than one stable base-paired structure. However, other alterna- tives have not been ruled out and we are preparing to determine the nucleotide sequence.

Previously, we suggested that the long inverted terminal repetition has a dual function (Garon et al., 1978; Wittek et al., 1980a). In addition to the pro- posed role in DNA replication, the inverted terminal repetition was shown to be transcribed and to code for early proteins. The arrangement of the early genes within the inverted terminal repetition and a detailed analysis of their transcription and translation products are presented by Wittek et al. (1980b).

Experimental Procedures

Preparation of Vacclnia Virus DNA A plaque-purified isolate of vaccinia virus (strain WR) was grown in HeLa cell suspension cultures and purified by sedimentation through a sucrose cushion and a sucrose density gradient essentially as described by Joklik (1962) except for the following: a Dounce homogenizer was used to disrupt cells; the cytoplasmic fraction and the resuspended pellet obtained after sedimentation through a 36% (w/v) sucrose cushion were digested wilhO.25 mg/ml of crystallized trypsin (Worthington) at 37°C for 30 min; sonication steps were avoided, and an SW252 rotor was used for sucrose gradient centrif- ugation. DNA was extracted from vaccinia virions as described (Garon et al.. 1978).

Preparation of XgtWes*A@ Recombinant DNA The construction and characterization of a hgtWES@ recombinant carrying the terminal Eco RI fragment (approximately 9000 bp) from vaccinia virus (WR) DNA have been described (Wittek et al., 198Oa). Phage was propagated In E. coli LE 392 (Enqutst et al.. 1979) and purified essentially as described by Hager et al. (1979) under Pt containment conditions. To isolate DNA from the phage suspension, EDTA and proteinase K were added to final concentrations of 20 mM and 100 pg/ml, respectively. Following incubation for 1 hr at 37°C. the reaction mixture was extracted repeatedly with phenol and then with chloroform. The DNA solution was then dialyzed against 50 mM Trts-HCI (pH 7.5). 50 mM NaCI.

formed in reaction mixtures recommended by the suppliers. Diges- tions were stopped by the addition of one fourth vol of 100 mM EDTA.

‘.12% Ficoll and 0.2% bromophenol blue.

Agarose Gel Electrophoresis Electrophoresis was performed in vertical slab gels using a buffer described by Loening (1967) with addition of 18 mM NaCl and pH adjustment to 8. Gels were stained with 1 fig/ml of ethidium bromide in electrophoresis buffer. DNA fragments were excised from agarose gels and purified by binding to glass powder (Vogelstein and Gillespie, 1979). For autoradiography, gels were dried onto DEAE-paper using a suction apparatus (BioRad). Agarose gels were blotted onto nitro- cellulose using the procedure of Wahl, Stern and Stark (1979) and “P-labeled probes were prepared by nick translation (Rigby et al., 1977).

Polyacrylamide Gel Electrophoresls Electrophorests was carried out in 10% polyacrylamlde slab gels in Tris-borate buffer (Peacock and Dingman, 1967) and stained with ethidium bromide. Autoradiographs were scanned with a soft laser densitometer (LKB) and areas under the curves were measured with a Numonics Graphic calculator.

End Labeling of DNA with Polynucleotlde Kinass The polynucleotide kinase exchange reaction was carried out in 100 pl reactions containing 25 mM imidaxole-HCI (pH 6.6). 45 mM KCI. 0.3 mM ADP, 10 FM ATP, 20 FCi of y-32P-ATP (2000 Ci/mmole) and 10 units of polynucleotide kinase, essentially as described by Berkner and Folk (1977) with Hinf I or Taq I digests of vaccinia virus DNA (2 pg). purified cloned DNA (0.125 ng) and @Xl 74RF DNA (1.25 cg). After 30 min at 37°C. the mixture was extracted with phenol and precipitated twice with ethanol.

End Lsbeting of DNA with DNA Polymerase Recombinant DNA was cleaved with Eco RI and the staggered ends were labeled with the large fragment of E. coli DNA polymerase I purchased from Boehringer-Mannheim. The 50 Fl reaction mixture consisted of 50 mM Tris-HCI (pH 7.6). 5 mM MgCl*. 1 mM DTT, 250 mM NaCl and 2.5 Fg of bovine serum albumin, 20 ng of Eco RI- cleaved DNA, 50 $i each of a-32P-dATP and u-~*P-TTP (>300 Ci/ mmole) and three units of DNA polymerase.

Partial Digestions of End-Labeled DNA Restriction sites in terminally labeled DNA fragments were mapped by the partial digestion procedure of Smith and Birnstiel (1976). Reaction mixtures contained approximately 10.000 cpm of end-la- beled DNA, 1 pg of carrier DNA and 0.5 units of restriction endonu- clease in a final volume of 80 Fl of the appropriate restriction buffer. At various times after the addition of the restriction enzyme, 16 pl portions of the incubation mixture were added to 4 FI of 100 mM EDTA. 12% Ficoll and 0.2% bromophenol blue. To obtain complete digestions, additional enzyme was added to the rest of the reaction mixture and the incubation was continued for 1 hr.

Acknowledgments

We wish to thank Bahige Baroudy for a sample of the purified 70 bp repeated fragment, Elaine Winters for carrying out a nick translation and blot hybridization and Jonathan Cooper for useful discussions. unpublished information and critical reading of the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 23, 19SO

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