Proc. NatL Acad. Sci. USA Vol. 78, No. 2, pp. 1090-1094, February 1981 Genetics A mechanism ofDNA transposition (bacteriophage Mu/transposable genetic elements/integrative replication/DNA amplification/chromosomal translocations) BRASIKA M. HARSHEY AND AHMAD 1. BUKHARI Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Communicated byJ. D. Watson, September 10, 1980 ABSTRACT Bacteriophage Mu and many other transposable elements undergo transposition by a process that involves replica- tion ofthe element. We describe here a mechanism by which such integrative replication maytake place. We have examined electron microscopically the DNA structures generated in host cells after Mu induction and have deduced the following steps in the trans- position process. (i) Association. A protein-mediated association is brought about between the transposable element and the target '4' 6 DNA. (ii) Attachment. One end of the element is nicked and at- tached to a site that undergoes a double-stranded cleavage. (iii) Roll in replication. While one strand ofthe target DNAis linked to the nicked strand ofthe element, the complementary strand ofthe target DNA is used as a primer for replication into the element such that the replicating DNA is threaded through the replication complex. (iv) Roi-in termination. When the distal end of the ele- ment arrives at the replication complex, replication is terminated. The roll-in replication mechanism can also explain laying down of tandem repeats-i.e., amplification ofcircular DNA sequences. It has become evident in the last few years that spatial arrange- ments of genetic material may undergo frequent changes both in prokaryotes and eukaryotes. Specific DNA sequences, called "transposable elements," are the primary mediators of such rearrangements (see ref. 1). Studies on bacteriophage Mu, a giant transposable element, suggested that the mechanism oftransposition may involve rep- lication ofthe element (2). The general model ofintegration de- veloped from these studies can be summarized in the scheme 1TE2 + 34 -. 1TE2 + 3TE4 in which 1TE2 is the donor host DNA containing the transpos- able element TE linked to sequences 1 and 2; 3 and 4 represent the recipient DNA sequences. The element appears in both products of the reaction-i.e., is duplicated during transposi- tion. The above scheme can also be written as FIG. 1. Interaction ofmini-Mu plasmids with host DNA. (A) Mini- Mu plasmid in contact with E. coli DNA. (B) Mini-Mu plasmid with 1TE2 + 34 -- 1TE4 + 3TE2. a tail (key structure). After Mu induction, total cellular DNA was extracted and prepared for electron microscopy by the Kleinschmidt Sequence 1 is now linked to sequence 4 and sequence 3 is linked technique (8). to sequence 2, and the element is present at each linkage. Note that the scheme is formally equivalent to reciprocal chromo- serve interactions between a transposon and its target sites on somal translocations, in-which 1 and 2 represent sequences on the host chromosome by electron microscopy using small plas- one chromosome and 3 and 4 are sequences on another. If se- mids containing internally deleted Mu bacteriophages (mini- quences 1 and 2 and sequences 3 and 4 are present on circular Mus) as model systems. We were able to show previously that DNA molecules, then the transposable element-mediated ille- these plasmids made physical contact with the chromosomal gitimate recombination between the two molecules will result DNA (8). We have also examined both the DNA structures gen- in fusion of the two molecules (replicon fusion). Such fused erated in Escherichia coli cells upon prophage Mu induction structures, called "cointegrates," have been observed with al- and those arising from infection by Mu particles. Based on our most alltransposable elements (5-7). observations, we propose here a mechanism for transposition in To study the process of transposition we have sought to ob- which the transposable element is replicated into a new site by roll-in replication. Certain features ofthis mechanism are simi- The publication costs ofthis article were defrayed in part by page charge lar to those proposed earlier (9). payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18.U. S. C. §1734 solely to indicate this fact. Abbreviation: kb, kilobase(s). 1090 Downloaded by guest on September 23, 2021 Genetics: Harshey and Bukhari Proc. NatL Acad. Sci. USA 78 (1981) 1091 IL~~~~~~~~~~~~~~~~~~~ D E~~~~~~~~~~~~~~ 01~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 FIG. 2. Electron micrographs ofkey structures after induction ofMu prophage. The size ofthe circles and the tails attached~to them vary consid- erably. Circle sizes: A, 2.6kb; B, 5.1 kb; C, 17kb; D, 29kb; E, 117 kb. Transposition intermediates and key structures standing the mechanism ofMu transposition. The number ofthe key structures is apparently increased by Pronase treatment and Bacteria lysogenic for Mucts62, a temperature-inducible mu- phenol extraction. The observation that the circular parts ofthe tant, and containing in addition a mini-Mu plasmid (8) were in- key structures can be smaller than one Mu length implies that duced by the standard procedures (10). The association between only one end of Mu attaches at a target site at one time. Yet we the mini-Mu plasmid and the E. coli chromosomal DNA, after know that both ends ofMu are required for full-scale-Mu repli- induction ofthe Mu prophage, was'investigated by electron mi- cation. Deletion ofone end drastically affects the replication of croscopy (8). We saw structures in which the plasmid seemed to Mu DNA (12). Furthermore, no freely replicating Mu DNA, be attached to the chromosome at a single point. We have also separate from the chromosomal DNA, can be seen. Based on seen structures in which the circle had a standard plasmid size these observations, we will divide the replication/integration but had a tail of an indeterminate length. We will refer to the cycle ofa mini-Mu plasmid into four discrete steps. The model structure in which a circle is attached to a tail as a "key struc- for transposition based on these steps is shown in Fig. 3. This ture. " These structures are shown in Fig. 1. mechanism of DNA transposition can explain the generation of Key structures are also readily seen when a Mu lysogen with- all DNA rearrangements associated with Mu as well as those as- out plasmid is induced. These have been reported (11). They sociated with some other DNA sequences as discussed below. consist of circles of all sizes and have tails of various lengths. Step 1. Association. It has been shown (13) that Mu DNA be- They are seen as early as 10 min after induction, and their num- comes associated with host DNA after infection and that this as- ber increases with time. Their presence is dependent on the sociation requires A and B proteins of Mu. Similarly, the func- products of genes A and B of Mu. The number of these struc- tioning ofat leastA and B genes is required for interaction ofthe tures is at least 5 times greater when the DNA samples are Mu-containing plasmids with the chromosome (8). Further- treated with Pronase and extracted with phenol forexamination, more, there is some evidence that the association also requires compared to the DNA samples observed without Pronase and active replication of host DNA (14). Thus, Mu proteins must phenol treatments. recognize the ends ofMu or sequences near the ends and must The lengths of the circles in these keys varied from 1 to 200 have affinity for some component ofthe replication complex of kilobases (kb) (Fig. 2). Ofthe 25 molecules examined, more than E. coli DNA. Either one end or both ends of Mu may be in- 50% had a circle size ofless than 38 kb, the length ofMu DNA. volved in bringing about this association. A possible explanation ofhow these structures arise is inherent Step 2. Attachment. Because mini-Mu plasmids always show to the mechanism oftransposition proposed here. single-point contact with host DNA, we postulate that attach- ment occurs at one end ofthe element. A simple way to achieve Mechanism oftransposition this is to have a single-stranded nick at one end ofthe element and a double-stranded break at the target DNA and to link the Because the key structures are generated as a consequence of nicked end ofthe element to one host DNA strand such that the Mu induction, we can assume that they are important in under- complementary host DNA strand provides a primer for DNA Downloaded by guest on September 23, 2021 1092 Genetics: Harshey and Bukhari Proc. Nad Acad. Sci. USA 78 (1981) Target + a R Donor 1. Protein-mediated association L R L R -k C O __ _ _ w~~~~~~~~~~~~~~~~~~~~~~~~pu _ _ _ 0 N N R L 2. Attachment b L L R 3. Roll-in replication -l N _ L 4. Roll-in termination FIG. 4. Cointegrate formation versus simple transposition. When the replication complex arrives at the distal end ofthe element, the 3'- end ofthe donor parental strand could be cut and ligated to the target strand (a). This would result in the insertion ofthe entire plasmid car- L rying the transposable element into the target site, generating a coin- tegrate structure. However, if the 3' end of the newly synthesized LIf strand is recognized for ligation (b), this would result in the simple transposition ofthe element into the target site, regenerating the orig- inal plasmid. FIG. 3. Mechanism of transposition. Arrowheads indicate 3'-hy- droxyl ends ofDNA strands; solid circles indicate 5'-phosphate ends. 1. Donor (ww transposable element) and target molecules are brought Step 3. Roll-in replication. Replication of the element then together by a protein that recognizes sequences at both ends of the proceeds by a roll-in mechanism on one strand by extending the transposable element and a sequence in the target molecule.