DNA intermediates in transposition of phage Mu

Cell, Vol. 29, 561-571,

June

1962,

Copyright

0 1962 by MIT

DNA Intermediates

in Transposition

Rasika M. Harshey, Ron McKay and Ahmad I. Bukhari Cold Spring Harbor Laboratory P.O. Box 100 Cold Spring Harbor, New York 11724 Summary Transposable genetic elements can insert into DNA sites that have no homology to themselves. Evidence that there is a physical linkage between a transposable element and its target DNA sequence during transposition comes from studies on bacteriophage Mu DNA transposition in which plasmids containing Mu DNA have been shown to attach to host DNA. We report the isolation of key structures, seen after induction of Mu DNA replication, after cloning lac operator into Mu DNA and using the lac repressor-operator interaction to trap Mu DNA on nitrocellulose filters. We have localized Mu sequences within these structures in the electron microscope by visualizing the lac operator-repressor interaction after binding with ferritinconjugated antibody. This analysis shows that key structures contain replicating Mu DNA linked to non-Mu DNA and that replication can begin at either end of Mu. Introduction Movable genetic elements, postulated to exist in maize (McClintock, 1950, 19561, were first discovered in bacteria in the form of insertion sequence elements, transposons and bacteriophage Mu. They have since been found in a variety of eucaryotes (see Cold Spring Harbor Symp. Quant. Biol. 45, 1980). They are involved in illegitimate recombination events in bacteria, which cause insertions, inversions and deletions, and have been implicated in the promotion of genetic changes in eucaryotes as well. They are also capable of modifying gene expression by insertional inactivation, through transcription termination or by promoting transcription (Bukhari et al., 1977). Although there are differences in the details of their DNA sequence, these movable genetic elements may share similar recombination mechanisms. Bacteriophage Mu offers a convenient system for studies on DNA rearrangements. When its transposition functions are induced, Mu goes through 100 transposition events per cell within one generation. This makes it possible to isolate and study the DNA intermediates in transposition. Although phage Mu is 36 kb in length, its entire genome is not necessary for transposition. Construction of mini-Mu (Mu DNA that has retained its extremities but has its central region deleted to various extents) has narrowed the sequences required for transposition to within 1 kb of the left end and 0.15 kb of the right end (Chaconas et al., 1980a; Coehlo et

of Phage Mu

al., 1980; Desmet et al., 1980; Howe and Schumm, 1980; Kamp and Kahmann, 1980). Functions of gene A, gene 6 and possibly gene arm (Waggoner et al., 1981) are required for transposition. That replication is necessary for transposition has been shown by studies on prophage Mu induction (Ljungquist and Bukhari, 1977) and has been supported by other studies (Chaconas et al., 1980a, 1980b, 1981 b). Involvement of replication during transposition has recently been inferred from studies on cointegrates or fused DNA structures, found originally in Mu (Toussaint and Faelen, 1973) and subsequently shown to occur with all transposable elements (Gill et al., 1978, for reviews see Calos and Miller, 1980, and Starlinger, 1980). Although multiple rounds of transposition occur during the lytic cycle, Mu DNA has not been found replicating in a form that is free of host DNA. Replicated copies of Mu are integrated at new sites in a manner that does not permit the appearance of freely diffusible copies in the cytoplasm. To understand how this is accomplished, we studied the DNA structures produced upon replication of Mu (Chaconas et al., 1980a; Harshey and Bukhari, 1981). SchrGder et al. (1974) reported the presence of circles with tails upon replication of Mu. In our study, we found key structures, which are circles of variable size attached to tails, also of variable size. Various schemes have been proposed to explain how replication of an element can result in its transposition (Grindley and Sherratt, 1978; Shapiro, 1979). On the basis of the DNA structures we observed upon Mu replication, we proposed a scheme for transposition (Harshey and Bukhari, 19811, which explained all the known DNA rearrangements associated with transposition (Figure I). Our scheme implicated the key structures as intermediates in Mu DNA transposition. To study these structures further, we allowed a minMu lac plasmid to undergo transposition and then trapped all mini-Mu-containing DNA on nitrocellulose filters by using the lac operator-repressor interaction. This technique enriched for key structures as well as for plasmids attached to host DNA. We have analyzed the key structures and mapped the position of Mu DNA within them. Using a technique described previously (Reed et al., 19751, we visualized in the electron microscope the lac repressor binding sites on the DNA by complexing the repressor with ferritin-conjugated antibody. The location of the bound ferritin cores was measured with respect to the junction between the circle and the tail in the key structures. We also carried out a restriction enzyme analysis of the key structures. Our data show that key structures contain replicating Mu DNA, supporting our previous proposal. These results also suggest that replication may begin at either the lefl or the right end of Mu DNA.

Cell 562

Figure

1.

A mechanism

of transposition

Transposable element tin ) and a target site are brought together by a protein that recognizes sequences at both ends of the transposable element (TE) and a sequence in the target B site (L and R define the ends of the TE). The target site then undergoes a double-stranded cut. Replication of the TE is initiated at one of its ends by placement of a nick in one strand, which is then ligated to the exposed S-phosphate of the target strand. The 3’-hydroxyl of the complementary target strand is used as a primer to extend into the TE (Solid circles indicate V-phosphate ends; arrowheads indicate 3’-hydroxyl ends. This assignment is arbitrary, and the strand polarities may well be the opposite of those indicated.) During replication, the free target strand is held in place by the replication complex. Replication is terminated when the other end of the TE passes through the replication complex and a second ligation event joins this end to the free target strand. (a) and (b) depict two possible situations depending upon the orientation of the initial ligation step. In (a) the completion of transposition would result in an inversion of DNA sequences between the two copies of the TE. In (b) the transposition products would be circles with one copy of the TE each, which would also represent deletions of host DNA sequences from either end of the TE. In both (a) and (b), disruption of the transposition complex during DNA isolation, which involves a protease-treatment and phenol-extraction step, would yield key structures (Harshey and Bukhari. 1981).

Results Purification of Transposition Intermediates by lac Operator-Repressor Interaction Since Mu DNA is always found linked to host DNA, it is difficult to isolate a well defined population of replicating molecules. We therefore cloned the lac operator sequence within mini-Mu (Chaconas et al., 1981 a) with the idea of using the lac operator-repressor interaction to trap all mini-Mu-containing DNA structures on nitrocellulose filters and hence purify them above the background of non-Mu DNA. Figure 2 shows the results of an experiment to determine the specificity of such a binding. A3’P-labeled mini-Mu lac plasmid, pCL198, was digested with Pvu II, bound to lac repressor and antibody and passed over nitrocellulose filters as described in Experimental Procedures. Bound DNA, eluted by IPTG, was subjected to electrophoresis on a 0.5% agarose gel. The gel was dried and autoradiographed. Lane 1 shows the Pvu II digestion pattern of pCL198. Lane 2 shows the DNA fragments retained on the filter after binding to lac repressor and antibody. The same amount of DNA was used in both lanes. Only one DNA fragment, D, is retained on the filter. This is the fragment containing the lac operator (Figure 2a). Recovery of this fragment is almost quantitative. Alternatively, the DNA can be eluted from the filters with 1% SDS. We raised monoclonal antibodies to the lac repressor, because we had been having trouble with retention of nonspecific fragments on the filters. We found subsequently that washing filters with sodium pyrophosphate eliminated this problem, and that regular antiserum could be

substituted for monoclonal antibodies with the same result. Use of antibody, however, greatly enhances the yield of the specific DNA fragment recovered from the filters. Using this technique, we purified all mini-Mu-containing DNA from a strain that carried the mini-Mu lac plasmid pGC404 that was undergoing active transposition after Mu induction. Examination under the electron microscope of the DNA thus purified revealed a considerable enrichment for free plasmid, for plasmids attached to the chromosome and for key structures. This suggested that key structures contain Mu DNA. Visualization of lac Operator-Repressor Complexes with Ferritin-Conjugated Antibody To map the position of mini-Mu in the isolated structures, we used a technique originally described by Reed et al. (1975) in their study of T-antigen binding to SV40 DNA. The lac operator sites on the DNA were visualized in the electron microscope after lac repressor was fixed on it with glutaraldehyde, then lac repressor antibody and ferritin-conjugated anti-antibody were bound and again fixed with glutaraldehyde. The complex was purified over a CLGB Sepharose column and spread for electron microscopy. The method was standardized with partially denatured pGC404. Under the denaturation conditions used, pGC404 shows a characteristic denaturation map, with a small denaturation bubble marking the left end of Mu and a larger one marking the right end (Figure 3~). The lac operator sequence is located 5 kb from the left end (Figure 3a). Figure 3c shows a

tp3rmediates

b

in Mu DNA Transposition

I

b

2

A,BC-

1

n Fractional

G-

Figure 3. Visualization mid pGC404

l-l length

of lac Repressor

Binding

to Mini-Mu

lac Plas-

(a) Schematic diagram of mini-Mu lac (pCL198) showing tne distribution of Pvu II sites. The thin line represents mini-Mu sequences where L and R are the left and right ends of Mu. The crosshatched area is the lac operon inserted within mini-Mu (Chaconas et al.. 1981 a). The kc operator sequence is carried within fragment D. (b) 32P-labeled pCLlg8 was digested with Pvu II, bound to lac repressor and monoclonal antirepressor antibody and then passed over nitrocellulose filters. Sound DNA was eluted from the filters with 1 mM IPTG and subjected to electrophoresis on a 0.5% agarose gel. The gel was dried and autoradiographed. (Lane 1) Pvu II digestion pattern before binding; (lane 2) Pvu II digestion pattern after binding to lac repressor and antibody. Only fragment D. which contains the lac operator, is retained on the filter.

(a) Schematic diagram of pGC404. The thin line represents mini-Mu DNA, and L and R its left and right ends. The crosshatched area represents the lac operon inserted within the Mu sequences, and (0) is the position of the lac operator sequence. tb) Plasmid pGC404 DNA was partially denatured then bound sequentially to lac repressor, to antibody to lac repressor and to ferritin-conjugated anti-antibody. The whole complex, fixed with glutaraldehyde, was spread for electron microscopy. The histogram shows an analysis of 30 such complexes. The distance of the ferritin core from the left end of mini-Mu is represented as a fraction of the total length of the molecule. The distance of the lac operator sequence from the left end is approximately one fifth the length of the whole plasmid molecule. (c) Electron micrograph of a pGC404 molecule showing the partially denatured left and right ends of mini-Mu, with an electron-dense ferritin core located at the position of the lac operator sequence.

pGC404 molecule that carries an electron-dense ferritin core at the appropriate position. Figure 3b shows data from 30 complexes where the distance of the ferritin core from the left end is represented as a fraction of the total length of the molecule. About 60% of the molecules had a ferritin core bound to them, and, as can be seen from the histogram, a significant proportion of these had the ferritin core in the expected position. In no case did we find more than one ferritin core per molecule. However, 6 of 30 ferritin cores (20%) were bound to incorrect sites. This is a substantial frequency of error. Since we had used partial denaturation mapping for determining the orientation of the molecules, it was possible that the error arose from the denaturation mapping rather than antibody specificity and that the four ferritin cores at position 0.8 were really at position 0.2. We therefore also performed the analysis after linearizing pGC404 with Xho I, which cut just outside the left end of Mu, or with Sma I, which cut just outside the right end, within pSC101. When Xho I was used for linearization, 36 of the 40 molecules analyzed were at a distance of 0.2 from one of the ends, and four were scattered

randomly over the remaining length. Essentially similar results were obtained with Sma I, with more than 90% of the cores mapping at a distance of 0.56 from one of the ends. We conclude from these results that the technique allows us to map mini-Mu sequences within the DNA structures we had purified over nitrocellulose filters. The experiment was repeated with DNA we had isolated from the transposition-proficient pGC404containing strain after purification over nitrocellulose for lac operator sequences. This DNA was complexed with repressor, antirepressor and ferritin-conjugated anti-antirepressor and examined as described above. More than 70% of the molecule8 seen had ferritin cores bound to them. The key structures were photographed, and the positions of ferritin cores were mapped (Table 1). The criteria used for end assignments primarily took into account the length of the mini-Mu, the position of lac operator site within it and the length of the circle and tail in the key structures. Binding of more than one ferritin core per key structure was considered significant. When only one ferritin core was bound per key structure, it was considered

Figure

2. Purification

of lac Operator

DNA

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Table

1. Analysis

of key Structures

Isolated

from BU7064:

Sizes

of Structures

and Position

of Ferritin

Cores

Location Ferritin Cores (kb from junction of circle and tail)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Circle tkb)

Tail (kb)

No. Ferritin Cores

12 9.4 10.6 35.5 6.9 65.1 41.5 4.7 22.1 20 5 7 40.2 14.9 37 9.2 15.6 12.9 14.5 24.6 16.2 25 6.6 11.2 25.5 12.9 3.0 24.0 9.8 14.7 17.5 28.0 25.0 11.2 13.9 16.5 22.7 8.8 48.9 56.1 80.2 27.0 51.5

97 55 83.5 9.7 31 22.2 53 11.5 14.9 13.5 32.7 45 5 22.5 10.7 20.5 22 35.6 38.0 35.0 24.0 36.0 31 .o 52.0 49.0 11.2 15.5 25.5 10.2 5.2 9.1 10.2 16.5 36.7 50.9 17.5 11.2 2.2 11.6 20.1 20.5 15.6 14.5

2

On Circle

On Tail

1; 1 at junction

2 2

0.4; 0.4 3

3 2

40; 10 4.5: 4.5

2

6.5 5.3: 5.3

3 8 10 9.2

10.5 8 2 2

8.7 1.2; 1.2 5.2

2

0.5 0.7

2

3.2

2 1

on Them

End at which Replication Started L L L L or R R L L or R R L R R

5.2

L L or R

2

L L

9.4 3.5

R L or R

3.7

3.4 7.0 4.8

L or R R L

2 2

6.7 1.8 5.2

7.1 1.6

L L or R

2

2.3 1.5

8.2

2

3

significant or not depending on the size of the molecule. In Table 1 molecules 9, 13, 18, 34, 35, 40, 42 were not assigned any end because they might have contained another operator site in the molecule that had failed to bind the repressor. Figure 4 shows some of these molecules and an interpretation of their state of replication. The molecules A, B and C contain ferritin cores at positions equidistant from the junction of the circle and tail in the key structure. Thus replication of Mu sequences has occurred, and the junction must be a replication fork. The circle in molecule A is 12 kb and that in molecule B is 20 kb. Since the size of the mini-Mu is 16.5 kb, and the lac operator sequence is located 5 kb from the left end, molecules A and B could have arisen only if replication had started at the left end of Mu. Molecule D, which has a

2.4 10.9 3.2 22; 1.8

1.7

L L or R

2.5

L or R

1.8

L or R

single ferritin core located on the tail, has a circle size of 8.9 kb and could have arieen only if replication had started at the right end. Some molecules, such as the one with a circle size of 35.5 kb shown in C, could have started replication at the left or the right end, and their origin of replication could not be identified. This molecule is different from molecules A, B and D in the orientation of the initial DNA ligation event (see Figure 1 b). Of the 43 molecules examined, 6 could be identified as having started at the right end, 11 at the left end, and the rest fell into the ambiguous class. It was clear, however, that in any key structure, only one end of Mu was involved in replication. The circles also vary from a theoretical minimum size (for example, Table 1, #2) to a very large size, suggesting that Mu can transpose to sites that are very close to its original

Intermediates 565

Figure

in Mu DNA Transposition

4. Visualizing

Mu DNA within

key Structures

Mini-Mu lac DNA undergoing active transposition was isolated and prepared for electron microscopy as described in the text. Molecules A, 6 and C are key structures that contain ferritin cores at positions equidistant from the junction of the circle and tail. Molecule D shows only one ferritin core bound to the tail. The schematic diagrams at the side of each micrograph show an interpretation of the state of replication of the mini-Mu (&vt ). L and R represent the left and right ends of mini-Mu. The diagrams have not been drawn to scale.

point of insertion as well as to those several kilobases away. It would have been desirable to cleave these molecules further with restriction enzymes, but we were unable to get complete digests of the DNA after purification over nitrocellulose or after ferritin-antibody binding. The results, however, show conclusively that key structures contain Mu DNA. In some of them, replication is clearly seen and can be interpreted to have started at either the left or the right end of Mu. Restriction Enzyme Analysis of Replicating Mu DNA Cleavage of DNA isolated when Mu is undergoing active replication ought to give some characteristic DNA structures. Cleavage by Eco RI, which cuts Mu twice-at 5 kb and at 23 kb from the left end-would give one middle fragment of 18 kb. If Mu were inserted into the host chromosome, then the left-end and the right-end fragments would not be 5 kb and 23 kb; their length would vary according to the next Eco RI cleavage site available in the adjacent host DNA. Figure 5 shows the consequences of Mu replication in a key structure when the replication starts from the left (L) end. If the replication fork does not reach the first Eco RI site at the left end, then one cut would be at 5 kb, and the other two would be in the host DNA

to the left. This would give rise to forked molecules with two unequal arms and a tail that would be less than 5 kb in length (Figure 5a). If the replication fork goes beyond the first Eco RI site, so that the fork is situated within the middle fragment (MI, then one would obtain forked molecules with arms of equal length, whose total length, from the tip of one arm to the end of the tail, is exactly 18 kb (Figure 5b). If the replication fork has gone beyond the second Eco RI site, then one would obtain forked molecules that have arms of equal length but a tail of variable length, since the tail length would depend upon the restriction site in the adjacent host DNA to the right. The length of the arms, however, would be less than 13 kb, the length of the right Eco RI fragment (R), if we assume that replication does not go past the Mu end (Figure 5~). Similarly, if replication started from the right end of Mu (not shown), then the right end fragment would generate molecules with arms of unequal length, whose tail would be shorter than 13 kb. The forked middle fragment would still be 18 kb, but the left-end fragment would give molecules with equal arms whose lengths would be less than 5 kb, but whose tail would be of a variable size. Thus the forked molecules from the middle fragment would not give any information about the direction of replication, but some of the others would.

Cell 566

Since it was not possible to digest the purified structures (the lac repressor complexes) with restriction enzymes, we digested the total DNA extracted from Bu2084,40 min after induction of Mu replication functions. The molecules were examined in the elec-

Figure 5. Pattern of DNA Molecules Structures with Eco RI

Produced

upon Digestion

of key

(M4 ) Mu DNA. (-) host DNA. L. M and R are the left, middle and right Eco RI fragments of Mu. The numbers 5. 18, and 13 are sizes of the fragments in kilobases. In the diagram, replication is shown to start at the left end (L) of Mu. (a), (b) and (c) represent situations where the replication fork is travelling from L to R and is within fragments L, M and R. respectively. See text for a more detailed explanation.

Figure

6. Electron

Micrographs

of Replicating

Mu Cleaved

tron microscope, forked molecules were photographed and their lengths were measured. Of 100 forked molecules measured, 46 had arms of equal length. Of these, 20 were 18 kb. Seven of these are aligned in Figure 6 to show the replication fork at various distances from the end. The size distribution of the molecules with arms of equal length is shown in Figure 7a. Table 2 lists the actual lengths of X and Y of these molecules. Table 3 lists the measurements of the three arms in molecules where all three were unequal. From all the molecules with arms of equal length examined, we identified 12 that had initiated replication at the left end, and nine that had initiated replication at the right end. From the molecules with arms of unequal length, we identified ten that had initiated replication at the left end, and 17 that had initiated at the right end. A similar analysis was performed with the restriction endonuclease Hind Ill, which also cleaves Mu DNA twice and produces an internal fragment of 25 kb. An analysis of 57 forked molecules showed that 27 had arms of equal length (Figure 7b). Of these, 14 were 25 kb in length, the size of the internal Hind Ill Mu fragment. From this analysis we could identify nine left-end and nine right-end replication forks (data not shown). Some molecules did not fit any category. These constituted 2% of the molecules analyzed after Eco RI cleavage and 5% of those analyzed after Hind Ill cleavage. Their possible origin is discussed below. We conclude from the above two sets of data that replication can start at either one of the ends of Mu.

with Eco RI

Total DNA was extracted from Bu2084 40 min after induction of Mu replication. The DNA was digested with Eco RI and examined in the electron microscope. The micrographs show seven forked molecules with equal arms whose total length (X+Y) is 18 kb. the size of the middle Eco RI fragment of Mu (see Figure 5). The molecules have been aligned to show the replication fork at various distances from the end.

Intermediates 567

in Mu DNA Transposition

Table 2. Analysis of Forked Molecules Digestion of Replicating Mu DNA

with Equal Arms after Eco RI

Uncleaved Circle (kb)

bL

x+yecu Figure 7. Size Distribution Produced upon Digestion Enzymes

of Forked Molecules with Equal Arms of Replicating Mu DNA with Restriction

(a) DNA was digested with Eco RI and mounted for microscopy as described in the legend to Figure 6. Of the 100 forked molecules measured, 46 had arms of equal length. Of these, 20 were 18 kb in length, the size of the middle Eco RI fragment of Mu. (n) Number of molecules. (b) As in (a). except Hind III was used to digest the DNA. Of the 57 forked molecules, 27 had equal arms. Of these, 14 had a size of 25kb. the size of the middle Hind Ill fragment of Mu.

Discussion “Controlling elements” was the term coined by 6. McClintock to describe the genetic elements that activated or terminated the activity of certain genes in maize. Her conclusion that genetic elements could move from one chromo8omal site to another has been corroborated by the discovery of insertion elements in bacteria (Taylor, 1963; Saedler and Starlinger, 1967; Adhya and Shapiro, 1969; Malamy, 1970). Transposable elements have been found to be natural components of chromosomes and extrachromosomal elements in a variety of organisms (see Cold Spring Harbor Symp. Quant. Biol. 45. 1980). Their ubiquity has prompted the viewpoint that they are selfish genes whose sole aim is to perpetuate themselves (Doolittle and Sapienza, 1980; Orgel and Crick, 1980). In spite

1’ 2’ 3 4 5 6 7 8 9 10 11 12 13’ 14. 15 16 17 18’ 19 20’ 21 l 22 23’ 24 25’ 26’ 27’ 28 29 30’ 31 32’ 33’ 34 35’ 36’ 37’ 38’ 39 40 41 42’ 43’ 44 45 46 47 48 49 50

6.4 12.6 10.0 4.3 4.8 7.8 9.4 12.8 3.5 7.0 12.6 7.5 16.0 7.0 8.0 2.0 8.6 9.4 19.6 9.6 4.2 10.3 17.4 6.6 8.0 5.5 15.4 7.0 il.5 16.8 5.4 14.0 7.0 3.2 13.2 6.4 5.8 1.5 2.5 4.4 4.0 2.4 3.2 4.5 2.5 4.2

11.7 5.5 12.0 6.4 4.3 15.0 5.0 3.4 11.6 10.0 9.7 1.2 5.3 10.6 12.6 6.0 8.6 10.2 14.6 9.8 9.0 2.3 8.5 8.5 7.6 0.7 11.3 6.0 9.0 2.8 7.2 6.4 1.3 9.2 4.5 11.4 15.0 5.0 8.1 8.8 6.2 16 13.2 5.0 6.0 7.0 12.0 12.0 10.2 11.2

3.7 11.3

1.9

End at which Replication Started

L R R L L L L or R R L L

R ? L L or R

? L or R

L or R

L L R

4.8

R R R L or R L or R L or R A

The two equal arms are designated X, and the tail is designated Y. l Denotes the 18 kb fragment. Uncleaved circles were not included in the analysis in Figure 7a.

of their ubiquity, however, they appear to have no autonomous existence. This suggests that they may not be able to perpetuate themselves without being linked to other DNA sequences. From a study of the DNA rearrangements they cause, it has become evident that the movement of transposable elements is generally coupled to their replication. The first such evidence came from studies on bacteriophage Mu

Cell 568

Table 3. Analysis of Forked Molecules with Unequal Arms Produced afler Eco RI Digestion of Replicating Mu DNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

X (kb)

Y (kb)

2 (kb)

End at which Replications Started

17.9 11.5 14.4 9.2 15.0 16.0 10.0 8.2 10.7 9.4 16.3 13.7 10.8 5.0 10.2 12.2 12.6 13.9 10.4 10.4 18.0 12.4 14.4 5.4 10.2 12.2 14.2 10.2 5.6 7.0 12.5 10.8 10.2 16.2 12.5 4.2 5.1 4.9 7.8 9.5 29.2 12.6 11.5 9.1 8.2 4.8 14.6 12.6 12.5 16.6 15.1 10.3 13.4 8.5

39.7 25.0 12.0 10.0 25.9 22.7 20.7 9.5 37.7 13.9 32.4 16.4 17.2 7.5 20.6 6.0 18.9 20.5 12.0 12.6 10.2 14.2 16.2 18.6 13.4 10.6 15.6 12.9 6.3 10.0 14.2 12.4 9.4 8.5 16.5 5.6 4.0 2.6 10.2 10.4 16.5 10.9 10.9 10.6 10.2 5.1 13.8 11.8 10.8 11.5 16.5 9.2 11.6 7.0

7.3 1.9 2.2 3.6 11.2 1.4 1.6 3.6 4.7 1.1 1.5 6.3 4.5 2.0 8.4 4.0 9.0 5.2 8.2 7.2 2.5 4.6 12.0 2.1 5.6 4.4 2.5 3.6 4.4 6.0 1.4 2.5 8.6 7.5 1.3 1.5 3.4 2.4 6.2 4.8 4.7 3.8 8.2 6.5 6.4 3.7 12.6 1.2 2.6 2.9 3.6 4.1 4.9 3.5

R L or Lor L or R L or L Lor L or L L or R L or L R L or R L or R R L or L or R L R L or L or L or L R L or L or R R L or L L L R L or L or L or R R R L R L or L or L or L or L or L or L

The three arms have been arbitrarily is the shortest arm of the three.

designated

R R R R R R R R

R R

R R

R R R

R R

R

R R R

R R R R R R

X, Y and Z, where

Z

(Ljungquist and Bukhari, 1977) and has since been corroborated by studies on other transposable elements (for review see Starlinger, 1980; Campbell, 1980; Yarmolinksy, 1980). The finding that most transposable elements form cointegrates (fused repli-

cons whose junctions are marked by a duplicated transposable element) has strongly supported the element-duplication theory for transposition. Various schemes have been proposed to explain the invasiveness of these elements, all of which involve a duplication of the element (for review, see Bukhari, 1981). To interpret DNA structures we observed upon Mu replication, we proposed a scheme (Harshey and Bukhari, 19811, which differed from those proposed previously (Grindley and Sherratt, 1978; Shapiro, 1979) in that replication was semi-conservative, and only one end of the element participated in invasion and recombination at a given time. Replication then proceeded across the element to terminate at the opposite end, where a second recombination event generated either a cointegrate structure or a simple transposition event. We implicated key structures as the DNA intermediates in Mu transposition, where an intrachromosomal event of transposition links replicating Mu DNA to different host DNA sequences, accounting for the variability in size of these structures. We report the isolation of replicating intermediates of mini-Mu lac, using a method based on lac operatorrepressor interaction. This procedure enriched for key structures and for plasmids attached to the chromosome. By tagging the lac operator sequence with repressor, antirepressor and ferritin-conjugated antiantirepressor, we have mapped the mini-Mu sequences within the purified key structures. We have found structures in which two copies of the lac operator sequence (and hence mini-Mu) are located symmetrically at the junction of the circle and tail of the key structure (Figures 4A, 4B and 40. These structures provide strong evidence that the key structures contain replicating Mu DNA, and that the junction of the circle and tail in the key structure is a replicating fork. Structures that contained a single ferritin core (Figure 4D) could be interpreted in some cases to have started replication at the left or right end of Mu. However, we cannot rule out the possibility that in these structures the presence of the lac operator at that site may have resulted from a previous transposition event and not from replication of Mu. We have inferred from the position of the ferritin cores on key structures and from the sizes of forked molecules produced after Eco RI and Hind Ill digestion of replicating Mu DNA that replication could begin at either the left or the right end of Mu. Wijffelman and van de Putte (1977) presented indirect evidence that replication in Mu was unidirectional, from left to right. However, our results show that it can start from either end, although we cannot rule out the possibility that the left-to-right direction may predominate under certain circumstances. If replication started with equal probability from either end of Mu, and if the extent of replication is random so that replication forks are found with equal probability at all points within the 38 kb Mu genome,

Intermediates 569

A

b

8

in Mu DNA Transposition

R Sk+-+ R CG-

Figure 8. Schematic Structures

Representation

of Recombination

between

key

Recombination between Mu sequences in the key structures can give rise to a circle with two tails, which appears like the eye of a replication bubble whose arms are unequal (a). or to a circle hanging by its tail (b). The length of the tail would not, in most cases, exceed the length of Mu. (c)Two circles connected by a tail. The length of the tail would not exceed the length of Mu. This structure would ariSe if replication had started at the left end in some molecules and at the right end in others.

then one would expect-for example, upon Eco RI digestion-that the majority of molecules with arms of equal length (18/18+5 or 78% if all replication started at the right end, and 18/l 8+13 or 58% if all started at the left end) would lie within the 18 kb middle fragment class, instead of the 43% observed. Similarly, 36% (13/36) should have unequal arms if all replication started at the right end, and 14% (5/ 36) if all replication started at the left end, instead of the 54% observed. A similar deviation from the expected numbers was found upon Hind Ill digestion. There are two possible explanations for the underrepresentation of the central Mu fragment. One possibility is that the mechanism of transposition generates this nonrandom distribution of forked molecules. For example, it is possible that there are sequences near the ends of Mu that slow the progression of the replication fork, resulting in a nonrandom rate of replication over the length of the molecule. Another possibility is that this nonrandom distribution is an artifactual consequence of our extraction procedures. For example, if branch migration occurs, it might lead to a selective loss of forked molecules of the middlefragment class, because the forks are entirely homologous, unlike the initiating fragments. Branch migration would also lead to a loss of the terminating fragments with arms of equal length. Even though we have only speculative explanations, this nonrandom distribution does not alter the main conclusion we draw

that replication can begin from these data -namely, from either one of the ends of Mu. We have not been able to fit 2%-5% of the DNA structures analyzed into any expected category. In our experience, such bizarre structures increase in number if the strain is Ret+ or if the DNA is isolated very late in the lytic cycle. These structures might arise as a result of intermolecular recombination events between key structures or as a result of inefficient termination of replication at late times, resulting in runaway replication into adjacent host DNA. Some consequences of intermolecular recombination are schematically represented in Figure 8. Such structures have been reported by Resibois et al. (1982). Our results conform to the prediction (Harshey and Bukhari, 1981) that key structures are replication intermediates in which one end of the element is activated to start replication. This does not rule out the possibility that occasionally both ends of Mu may be activated to start replication simultaneously. However, such an event, if it occurs, must be infrequent. The results presented here are most easily explained by the scheme we proposed previously (Harshey and Bukhari, 1981). However, other schemes also can be adapted to our results. For example, if both ends integrated simultaneously, as proposed by Shapiro (19791, but replication only started at one end, accidental breakage of one DNA chain at the nonreplicating end would generate a key structure. We can, however, rule out schemes that propose diffusible copies or single-stranded intermediates in transposition (for review see Bukhari, 1981). Our findings support transposition schemes in which ends of elements invade and get linked to nonhomologous DNA sites to insert copies of themselves. Experimental

Procedures

Bacterial Strains and Plasmids The strains used were E. coli K-12 derivatives Bu2084 (A prolac met-, Sm’. recA Mucts62) and Bu7064 (Aprolac. met-. SmR. recA, Mucts62, pGC404-/ac+ 7~~). Plasmids pGC404 and pCL198 are mini-Mu plasmids containing the lac operon between the two ends of Mu that can transpose when supplied with transposition functions from a helper Mu prophage (Chaconas et al., 1981 a, 1980b). Media and growth conditions have been described (Bukhari and Ljungquist, 1977). Replication of Mu was induced by shaking cultures at 43’C until complete lysis had occurred. Bu7064 was grown in the presence of tetracycline (10 pg/ml). Biochemical Procedures The lac repressor and antibody raised in rabbit to lac repressor a gifl from M. Pfahl and P. H. von Hippel. Ferritin-conjugated antirabbit IgG was purchased from Appel Labs.

were goat

Cleavage Reactions with Restriction Endonucleases Eco RI endonuclease reactions were carried out in 0.1 M Tris-HCI buffer (pH 7.5). 6 mM MgCI1. 6 mM /3-mercaptoethanol, 50 mM NaCI. at 37°C for 2 hr. and Hind Ill endonuclease reactions in 6 mM TrisHCI (pH 7.4), 6 mM M&I., 50 mM NaCI. at 37’C for 2 hr. Purification of kc Operator DNA on Nitrocellulose Filters The repressor-operator binding assay performed was a modification of the one described by Riggs et al. (1970).

Cell 570

DNA and lac repressor were incubated in the binding buffer (0.01 M magnesium acetate, 0.01 M KCI. 1O-’ EDTA, 1 O-’ M dithiothreitol, 0.01 M Tris-HCI [pH 7.41, 50 Fg/ml BSA) for 10 min at room temperature. Monoclonal antibody to lac repressor was added, and incubation was continued for another 5 min. The mixture was then filtered dropwise through a prepared sheet of nitrocellulose (see below). Filters were washed three times with 10 ml each of 20 mM sodium pyrophosphate. This step was essential in eliminating all nonspecific binding. DNA remaining on the filter was then eluted with a solution containing isopropyl /3-thiogalactoside (1 mM in 10 mM Tris-HCI and 1 mM EDTA [PH 8.51) as described previously (Potter and Dressler. 1979). The filters (Schleicher and Schuell. BA85) were prepared for the binding assay as described by Potter and Dressler (1979) except that calf thymus DNA was eliminated from the final soak. Preparation of Monoclonal Antibodies We used lac repressor in complete Freund’s adjuvant to immunize female BALB/c mice on two occasions separated by 1 month. After another month, the mice were given a dose (-5 pg) of repressor by intravenous injection. Three days later the spleen was removed, and the dissociated cells were fused with the X83, NS-1 cell line according to the procedure of Galfre et al. (1977). Hybrid cell line supernatants were screened by an immunoassay for DNA binding proteins (McKay. 1981). Visualizing Protein-DNA Complexes in the Electron Microscope with FerritikConjugated Antibody The procedure used was essentially as described by Reed et al. (1975). with a few modifications. DNA was incubated with lac repressor in binding buffer for 10 min at 37°C. The binding buffer contained 20 mM sodium pyrophosphate as well. The mixture was chilled to 0°C. Glutaraldehyde in binding buffer was added to a final concentration of 0.1%. After incubation at 37°C for 10 min, antibody to lac repressor was added, and afler 10 min ferritin-conjugated goat antirabbit antibody was added. After an additional 10 min, the mixture was chilled to 0°C glutaraldehyde was added to a final concentration of 0.1% and the mixture was brought to 37OC for a final 20 min. The sample was passed over a small CL8B Sepharose column, which had been equilibrated in 0.01 M TrisHCI and 1 Om3 M EDTA (pH 8). Partial Denaturation of DNA for Electron Microscopy This procedure was carried out as described by lnman and Schnds (1970). Electron Microscopy Samples were mounted for electron microscopy with 40% formamide (Davis et al., 1971). Grids were rotary-shadowed with platinumpalladium, then examined and photographed with a Philips EM201 C electron microscope. Negatives were projected with an Omega ProLab enlarger, and the contour lengths of molecules were measured with a Numonics planimeter. Acknowledgments We thank M. Pfahl and P. H. von Hippel for their generous gift of lac repressor and lac repressor IgG. This work was supported by grants from The National Science Foundation and the National Institutes of Health. 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

December

14. 1981;

revised

March

22, 1982

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