DNA structures induced by mini-Mu replication

VIROLOGY

117, 320-340 (1982)

DNA Structures Induced by Mini-Mu Replication A. RESIBOIS,’ Laborattire

de Ghnktique,

Fact&

A. TOUSSAINT,

d.es S ciences,

Received July

Universiti

AND M. COLET

libre de Bruxelles, Rhode St. Gen&e, Belguim

accepted October

31, 1981;

29, 1981

The entire DNA of an induced Escherichia wli strain lysogenic for a mini-Mu able to replicate has been extracted after 50 min growth at 42” and examined with the electron microscope. Several unusual DNA configurations were identified: circles, keys, pending keys, dumbbells, asymmetrical “replication forks,” and inverted “replication forks.” The importance of these structures is discussed with respect to the different models proposed in the literature for the replication of Mu. INTRODUCTION

During the last few years the replication forms of several bacteriophages and viruses have been visualized by electron microscopy. The isolation and spreading of replicating forms has revealed processes as different as the rolling circles of phage X (Weissbach et aL, 1968)or 4x1‘74 (Koths and Dressler, 1978), the linear replicating intermediates of T7 (Wolfson et al, 19’71), the branched circles of P2 (Schnos and Inman, 1971), the branched concatemers of T4 (Mosig et aL, 1978), the circular forms of Py (Hirt, 1969), and the single-stranded intermediates of some adenoviruses (Lechner and Kelly, 1977) and f#&l (Inciarte et ak, 1980). The study of the replication of bacteriophage Mu is very complicated due to the fact that the phage DNA, which is convalently linked to the host DNA even in the viral particles (Daniel1 et ak, 1973; Bukhari et a& 1976) is most probably integrated in the Escherichia coli chromosome during the entire lytic cycle (Schroeder and Van de Putte, 1974; Razzaki and Bukhari, 1975; Ljungquist and Bukhari, 1979). This is the most likely explanation as to why the isolation of replicating intermediates containing only Mu DNA has never been successful. Schroeder et a& (1974) isolated four dis’ To whom reprint

requests should be addressed.

Crete species of DNA from an induced E. coli strain lysogenic for Mu: mature linear phage DNA, linear DNA which sedimented faster than the first one, circular relaxed circles with or without tails, and superhelical rings. Each of these classes contained both host and phage DNA. Particular attention was given to the circular molecules. Heteroduplexes made between the covalently closed circular DNAs and the linear Mu phage DNA confirmed the presence of the viral genome in the circles (Waggoner et al, 1974,1977). In 1976, using genetic experiments, Parker and Bukhari showed that each F’ episome generated in an induced Hfr strain lysogenic for Mu contained at least one copy of Mu. From this observation they concluded that the heterogeneous circles were formed by a process which directly involved Mu integration. Waggoner et al. (1977) concluded that at least one round of phage Mu DNA synthesis is necessary before closed circular DNA molecules can be detected in a CsClEtBr density gradient. The length of the circular molecules varies from 22 to 201 kb and is thus in no way related to the length of Mu. No homology could be found between the different host sequences covalently linked to Mu among the circles. The circles with a tail described by Schroeder et al. (1974) could be correlated with structures expected in a rolling circle mode of replication (Gilbert and Dressler,

323

0042-6822/82/040329-12%02.00/O Copyright All rights

Q 1982 by Academic Prees. Inc. of reproduction in any form reserved.

330

RlbXBOIS,

TOUSSAINT,

1968). However, these tailed circles exhibit a wide range of sizes which cannot be grouped in classes. They contain host DNA and their replication would lead to simultaneous amplification of Mu as well as adjacent host DNA. Waggoner and Pato (1978) have shown that the host DNA adjacent to a Mu prophage is not replicated at early times after induction. This, however, does not exclude the possibility that the bacterial DNA in the tailed circles is replicated. Harshey and Bukhari (1981) showed that Mu generates circles with a tail (key structures) in which the circle is as short as 1 kb. These key structures appear very early after induction (about 10 min). These authors proposed a model for Mu replication in which integration and simultaneous replication of the phage DNA induced formation of both keys and closed circular structures. It has been conclusively shown that the appearance of keys and circles is related to Mu replication. However, whether they are true replication intermediates remains to be demonstrated. These structures represent at most 2% of the total DNA. Besides replication intermediates they could also represent end products of the replication process or transposition intermediates. It is not known whether keys are the only unusual configuration which arise as a result of Mu induction. In an attempt to better characterize the structures involved in Mu replication-integration we chose to use a deleted Mu derivative, mini-MuA (RQsibois et aL, 1981) which is 10 kb long. This mini-Mu replicates almost as well as wild-type Mu (Waggoner et al, 1981) and carries an internal deletion covering all lysis and maturation functions so that it is unable either to lyse the host bacterium or to mature. Using such a mini-Mu we hoped that by lengthening the time of incubation after induction we would enhance the number of significant configurations generated by MuA replication and transposition and therefore have a better opportunity to observe structures different and even rarer than circles and keys.

AND

COLET

MATERIAL

AND

METHODS

Strains. Bacteria: MXR/RP$ MXR/ RP4::MuA26; MXR is recA, galE, Apro, lac (Faelen et al, 1978); RP4 was described by Datta et aL, (1971); RP4::MuA26 was isolated by transposition of MuA into RP4 (Faelen, unpublished result). Bacteriophages: MuA26; Muctsbz deleted from C to S (RQsibois et ah, 1981). DNA extraction. Overnight cultures of MXR/RP4::MuA26 were diluted 100 times in 20 ml fresh LB (Miller, 1972), grown at 30” with aeration to a concentration of about 2.108 bacteria/ml, shifted to and grown at 42” with vigorous shaking for 50 min. The cultures were centrifuged for 10 min in an SS34 rotor (Sorvall RC5 centrifuge) at 10,000 rpm. The pellets were washed twice with TE (Tris-HCl 10e2 M, pH 8, EDTA 10m3 M, pH 8) and resuspended in 1 ml of spheroplast forming mixture. The spheroplast-forming mixture contained 10 mg lysozyme, 1 mg RNase, and 10% sucrose (w/v) in 10 ml TES (5.10d2 M Tris-HCl, 5.10e2 M NaCl, 5.10V3 M EDTA, pH 8.5). After 10 min on ice, the cells were lysed by the addition of 0.5 ml sarkosyl2%, incubated at 0” for 15 min, and treated with proteinase K (0.06 ml of a solution at 5 mg/ml in TES) for 30 min at 37”. The DNA suspension was dialyzed for 2 hr at 4” against TE (pH 8.5), extracted twice with phenol equilibrated with TES, and dialyzed for 48 hr against TES. Electron microscopy. The whole induced E. coZi::MuA26 DNA was mixed with the Tris-EDTA-formamide mixture according to Sharp et al. (1972) and spread. Some of the DNAs were partially denatured before spreading in a mixture containing 6 M sodium perchlorate and 2% formaldehyde (Bade et aL, 1977). Only the measurements of the whole structures are reported here. The results of the partial denaturation will be reported elsewhere. The DNA was then collected on parlodioncovered copper grids, stained with uranium acetate, shadowed with 5-10 mg uranium oxide, and examined with a Siemens Elm 1A microscope at 8000 direct

*

*-

FIG. 1. (a) Closed circular nicked molecule; (b) key structure. The joint between the circle and the tail is indicated by an arrow (~40,000). FIG. 2. Key structures. (a) In this key, the very small circle measures less than 1 kb (~40,000); (b) large circle with a small single-stranded region at the junction between the circle and the tail (X42,000). 331

332

RESIBOIS,

TOUSSAINT,

AND

TABLE MEASUREMENTS

Free

1

OF THE LENGTHS OF THE DIFFERENT DNA THE INDUCED E. Cdi::MIN14lUa

keys

Pending

keys

Circles

Circle

Tail

Circle

Tail

15.9 21.5 22.9 25.8 48.2 54.1

1.3 1.9 4.8 11.6 21.6 26.0 26.7 27.8 31.3 32.1 39.1 53.7 56.0 59.8 65.0 65.8 62.1 89.6 119.6 121.1

>63 48.4 >35 >16 29.1 14.2 >39 >40 16.6 7.3 5.7 >60 265 6.8 15.0 SO >53 15.9 216.7 >36

0.8 1.5 2.8 4.7 10.7 13.3 25.4 32.1

3.8 8.4 0.4 0.5 5.1 9.8 9.6 8.2

D All the lengths of length.

COLET

are expressed

in kilobases

STRUWIJRES

Dumbbells Circle 17.3 19.2 19.7 33.1

(kb).

1

Circle 20.2 47.3 104.9 72.7

Double-stranded

magnification. The measurements were made with a Hewlett-Packard digitizer using double-stranded &X174 as standard of length (5.4 kb).

2

Join. 6.3 0.2 8.9 26.0

$X DNA

Several different DNA structures are observed in the DNA of the induced lysogen. Among them, only circular DNA molecules are sometimes seen in the control DNA extracted from MXR/RPI. 1. Closed Circular

Molecules (Fig. 1)

There are numerous closed circular molecules relaxed or supercoiled, free or apparently attached by one point to linear “bacterial” DNA. As described by others, their length is variable (Table 1) but they are always longer than the length of one mini-Mu. In the MXR/RP4 strain, super-

Invert. forks 5.1 7.2 8.3 8.9 20.9 23.9

was used

IN

Asymmet.

forks

Long arm

Short arm

34.6 22.1 137.2 196.2 35.6 85.4 16.8

9.9 10.9 108.3 7.10 17.7 19.75 9.6

as internal

standard

coiled circular molecules are seen rarely: their size is more uniform, about the 50 kb expected for the RP4. 2. Kw Structures

RESULTS

OBSERVED

(Figs. 1 and 2)

Next to the circles, the key structures are by far the most frequently seen structures. Out of 20 keys measured (Table 1) the shortest circle measures 1.3 kb and the longest one 121 kb. Most of them are longer than one mini-Mu and there is no apparent correlation between the lengths of the circles and the length of the miniMu. The tails are also verv variable in length and this length does not seem to be correlated either with the size of the miniMu or with the size of the circle. Most of the tails seem to be simple linear DNA, sometimes so long that it was impossible to measure them up to their end. As shown in Fig. 2, a short single-stranded DNA seg-

MINI-MU

REPLICATIVE

DNA STRUCTURES

333

ment is frequently visible at the junction between the circle and the tail; it could represent the delayed DNA synthesis of the lagging strand of a replicative fork. 3. Pending

Keps (Fig. 3)

Some keys are attached by their tails to long stretches of linear DNA. The circles in these keys are usually relatively small (0.8 up to 32.1 kb) and have a length which does not seem to be correlated to the length of the mini-Mu (Table 1). On the contrary there seems to be a relationship between the respective lengths of the tail and the mini-Mu since no tail longer than the length of the mini-phage DNA was ever observed (Table 1). 4 Dumbbells (Fig. 4) FIG. 3. Pending key. (a) The circle; (b) the very short tail; (c) the linear DNA to which the tail is attached (X44,000).

In this configuration two elosed circular DNAs are attached to the same tail, one at each extremity. The length of the linear

FIG. 4. Dumbbell. Two large circles of DNA (a) (b) are joined together segment (arrow) (X46,000).

by a very short DNA

334

RfiSIBOIS,

TOUSSAINT,

AND COLET

6. Asqm&icd

“~Replicution Fork” (Fig. 6)

At first glance these structures resemble a true replication fork. However, the difference in length of the two arms is so large that it cannot be the result of imprecision in the measurements (Table 1). There is a tremendous variation in the length of the strands within the “fork” and the length of the mini-Mu does not seem to be correlated either with the lengths of the arms or with the difference of length between them. Short segments of single-stranded DNA are sometimes visible in either of the arms of one or both forks. DISCUSSION

FIG. 5. Inverted “replication fork.” A central double-stranded DNA the length of which is less than 10 kb (a) with diverging points at both extremities (arrows) (X46,000).

DNA which joins the two circles is usually, but not always, shorter than the mini-Mu (Table 1). These structures are very rare and the number of molecules which we were able to measure was too small to allow a clear systematization. 5. Imerted

“Replicatian

Forks”

The use of the replicating mini-MU26 for the observation of Mu replication intermediates allowed us to observe rarer configurations such as dumbbells, pending keys, asymmetrical “replication forks,” and inverted “replication forks” in addition to the circles and keys described by Schroeder et al. (1974), Waggoner et al. (1974, 1977), and Harshey and Bukhari (1981). At first glance it seems impossible to find a correlation between so many different configurations (see Fig. 7). However, the observed structures can be characterized into four classes.

(Fig. 5)

Structures in which two double-stranded DNA segments join to make a single one which splits again after a certain length were termed “inverted replication forks” because a true replication fork appears like an eye structure in which a unique double-stranded DNA opens in two branches which rejoin after a variable length of parallel course. This term is only descriptive and does not imply that such a structure is really replicative. As shown in Table 1 in some of these structures the unique joining segment is shorter than a mini-Mu while in others it is longer. A single-stranded DNA region is sometimes visible at one or both junction points; it was never seen in the joining segment.

1. The Free Circles

As expected from previous observations (Schroeder et aL, 1974, Waggoner et al, 1974, 1977), we found that the free circular structures which consist of doublestranded DNA vary in size and that the sizes of the circles are not correlated with the length of the viral genome. These circles could represent an end product of Mu replication or transposition within which no process was occurring at the time of DNA extraction. All the models proposed for Mu replication predict the formation of such circles (Faelen et al, 1975; Shapiro, 1979; Arthur and Sherratt, 1979; Harshey and Bukhari, 1981).

MINI-MU

REPLICATIVE

DNA STRUCTURES

335

FIG. 6. Asymmetrical “replication fork.” The two arrows indicate the structures which could be similar to the growing points of a replication fork. However, between them, the two branches are very different in length since (a) is nearly two times longer than (b) (X44,000).

2. Free Keys The model proposed for Mu replication by Harshey and Bukhari (1981) derives directly from the observation of key structures. According to these authors, the key structure would be generated by the insertion of one end of one Mu strand at a staggered cut at the target site located at some random site within the bacterial DNA (Fig. 8a). This would create a replication complex which would proceed semiconservatively, generating two daughter genomes located both in the circle (Fig. 8~) or one in the circle and one in the tail (Fig. 8d), depending on whether the nicked Mu extremity had bound to the proximal or the distal nick of the staggered cut at the target site (Grindley and Sherratt, 1978). The keys which we observe fit well within this model: as expected their circles are sometimes shorter than the mini-Mu and their tails are often

lost in the total E. co2i DNA as postulated by the model. In addition we often see single-stranded segments located at the junc-

x.x_:-w rrl 7-Y

\

/

FIG. 7. Schematic drawing of the different configurations which were observed. 1, circle; 2, key; 3, pending key; these could result from the breakage of one circle in the dumbbell structures; 4, dumbbell; 5, inverted replication fork; these could result from breakage of the circles in the dumbbell and the ribosome-like structures; 6, asymmetrical replication fork; 7, ribosome-like structure.

\ 0

RESIBOIS, TOUSSAINT,

336

C

AND COLET

b

‘s

M

N

1

\

p,

12

a

FIG. 8. Formation of key structures according to the model proposed by Harshey and Bukhari (1981). (a) A nick has occurred at Mu c-end-staggered cuts (five base pairs apart in the case of Mu) indicated by the arrows 1 and 2 occur at the target site. This generates the structure shown in (b) where the free 3-‘OH ends at the target have been brought near the nicked Mu c-end and artifically dissociated to make the drawing clearer. (c) The 3’ end at the target, labeled 1 in (b) binds to the 5’ Mu c-end; this generates a key where the Mu lies in the tail at the circle-tail joint. (d) The other 3’ end, labeled 2 in (b), binds to the 5’ Mu c-end generating a key where the Mu lies in the circle at the circle-tail junction. Wavy lines represent Mu DNA, filled squares represent the target site, normal lines represent host DNA. Arrowheads indicate 3’-hydroxyl ends; solid circles indicate 5’-phosphate ends; C and S represent Mu c- and S-ends, respectively. MN0 and P are bacterial sequences flanking the Mu prophage and the target site.

tion between the circle and the tail, either in the circle or in the tail, supporting the suggestion that a replicative process is at work. 3. Pending Keys, L?umbbelLs, and “inverted Forks” in Which the Jtining Segment Is Shorter or Equal to the Length of the Mini-Mu Figure 7 summarizes the different configurations which we observed with the EM. It is probably easier to see in this schematic drawing how the three structures of this class are related. What we call an “inverted fork” could certainly be part of a pending key in which the circle was too big to be either conserved or mea-

sured, or part of a dumbbell with two giant circles. A pending key might be a dumbbell structure with one giant circle. All the tails of the pending keys and nearly every joining segment in the dumbbells and the inverted forks which we measured are shorter than the 10 kb of the mini-Mu genome. If this turns out to be a general feature it would be a very strong argument supporting another type of model, proposed for both Mu and transposons by Shapiro (1979) and Arthur and Sherratt (1979). In this model (Fig. 9), the two extremities of the transposable element are simultaneously niCked and inserted in a staggered nick at the target site. This creates a double replication analog and replication can then proceed from

MINI-MU

REPLICATIVE

DNA STRUCTURES

337

d FIG. 9. Generation of dumbbell, ribosome-like, pending key, and inverted fork structures according to the model proposed by Shapiro (1979). (a) The arrows indicate sites where nicks occur (at the ends of Mu and at the target site) to generate the structure shown in (b). (c) The 3’ end 1 at the target binds to Mu c-end, while the 3’ end 2 binds to Mu Send. This generates a dumbbell structure with Mu connecting the two circles. (d) The 3’ end 1 binds to the Mu S-end while the 3’ end 2 binds to the Mu c-end generating a ribosome-like structure where Mu connects the two circles. Breaks in both circles of either the dumbbell or the ribosome-like structure will generate inverted forks. Break in one circle of the dumbbell will generate a pending key. Break in one circle of the ribosomelike structure will generate an asymmetrical fork where one arm consists of Mu while the other contains host DNA. DNA structures and polarities are represented as in Fig. 8.

these replication forks through the inserted element. This model predicts the formation of inverted forks with an unreplicated central region the length of which cannot exceed the length of the transposable element. These inverted forks would be expected to be part of two types of structures: (1) dumbbells if each nick of the transposable element is joined to the nick of the target site situated on the complementary DNA strand (Fig. 9c); (2) “ribosome-like” structures (Fig. ‘7) if the ligation joins nicks of the transposable element and the target situated on the same DNA strand (Fig. 9d). These last configurations are very rare in our preparations but they are nevertheless present.

4. Inverted Forks with a Common. Stem Longer Than 10 h% and Asymmetrical F@?-h% Paolozzi et al (1978) suggested that Mu integrates preferentially at the bacterial replication forks. The same conclusion was drawn by Fitts and Taylor (1980) from mating experiments in which a synchronized culture of an Hfr strain was infected at different times with Mu and mated with a Mu nonimmune recipient strain. The time of entry of Mu in the recipient cell, monitored by zygotic induction, suggests that the location of the Mu in the Hfr chromosome is correlated with the position of the replication fork. If a transpositionreplication event of the type suggested by

RESIBOIS, TOUSSAINT,

AND COLET

1 FIG. 10. Generation of asymmetrical forks according to the model proposed by Harshey and Bukhari (1981). (a) The Mu prophage as well as the target site lie in a region of the bacterial chromosome which has been replicated. Nicks occur as in Fig. 8, at the sites marked by the arrows to generate the structure shown in (b). (c) The 8 end 2 at the target binds to the nicked Mu c-end, generating an asymmetrical fork where one arm contains host DNA (0’) and the other arm contains Mu and a longer segment of host DNA (0 + N). (d) The 3’ end 1 at the target binds to the nicked Mu c-end generating an asymmetrical fork where one arm consists of only host DNA (M) while the other contains the second Mu copy flanked by host DNA sequences (M and N). DNA sequences and polarities are represented as in Fig. 8.

Harshey and Bukhari takes place just in front of a host replication fork it will generate a pending key in which the tail represents the distance between the circle and the fork. On the other hand, the same type of transposition-replication event could take place just beyond the front of the host replication fork (Fig. 10). If one Mu extremity located in a newly synthesized

DNA strand is nicked and integrates at a staggered nick situated on the opposite daughter strand it will generate an asymmetric replication fork (Fig. 7). One arm of the fork represents the DNA segment located between the extremity of the bacterial fork and the target site, the other, the length of the DNA segment located between the fork and the nicked Mu ex-

MINI-MU

REPLICATIVE

tremity. One arm is always longer than the length of the mini-Mu, and the other can have a variable length. The structures we have described are thus compatible with both or one or another type of model. It is therefore possible that Mu replication-transposition occurs either by a process involving simultaneously both ends of the Mu genome and both sides of the target site (Shapiro’s, Arthur-Sherratt’s type model) or by a process involving linkage of one end of the Mu genome to the target site prior to replication, the second end being linked only after completion of replication (HarsheyBukhari’s type model). It can not be excluded that both type of events could take place at the same time or in succession during the lytic cycle of Mu. In relation to this it should be noted that we used a mini-Mu and an abnormally long time of induction. These conditions could enhance specifically configurations which are rarer or even absent after induction of a wildtype Mu under more physiological conditions. Long stretches of single-stranded DNA were not observed, a fact which does not support the idea of an eventual singlestranded intermediate in the replication of Mu. In addition, we never observed a true replication fork with replicated arms smaller than 10 kb, which eliminates the possibility of a Mu replication preceding excision and reintegration. In order to further correlate the observed structures with the different models of transposition-replication, it is necessary to locate the MuAZ6 within them. This is currently in progress using partial denaturation of the DNA used in this study and a partial denaturation map of MuA26. ACKNOWLEDGMENTS We thank R. Clayton for her critical reading of our manuscript and 0. P. Doubleday who gave us the $XDNA. This work was carried out under an agreement between the Universite libre de Bruxelles and the Belgian Government concerning priority actions in collective basic research and with help from the Fonda National de la Recherche Scientifique (FNRS). A.T. is chercheur qualifie from the FNRS.

DNA STRUCTURES

339

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SHAPIRO, J. A. (1979). Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements. Proc Nut. Acad Sci USA 76,1933-1937. SHARP, P. A., Hsu, M. T., OHTSUBO,E., and DAVIDSON, N. (1972). Electron microscope heteroduplex studies of sequence relations among plasmids of E. coli. I. Structure of F-prime factors. J. Mel Bid 71, 471-497. WAGGONER, B. T., and PATO, M. L. (1978). Early events in the replication of Mu prophage DNA. J. Viral 27,587-594. WAGGONER, B. T., GONZALES, N. S., and TAYLOR, A. L. (1974). Isolation of heterogeneous circular DNA from induced lysogens of bacteriophage Mu1. Proc Nat. Acad Sci USA 71,1255-1259. WAGGONER, B. T., PATO, M. L., and TAYLOR, A. L. (1977). Characterization of covalently closed circular DNA molecules isolated after bacteriophage Mu induction. In “DNA Insertion Elements, Plasmids and Episomes” (A. I. Bukhari, J. Shapiro, and S. Adhya, eds.) pp. 263-274. Cold Spring Harbor Press, Cold Spring Harbor, N. Y. WAGGONER, B. T., PATO, M. L., TOUSSAINT, A., and FAELEN, M. (1981). Replication of mini-Mu. V&Zogy 113.379-387. WEISSBACH, A., BARTL, P., and SALZMAN, L. A. (1968). The structure of replicative X DNA. Electron microscope studies. Cold Sprint Harbor Symp. Quant. BioL 33,525-531. WOLFSON, J., DRESSLER, D., and MAGAZIN, M. (1971). Bacteriophage T7 replication: A linear replicative intermediate. Proc Nat. Acad Sci USA 69, 499507.