La postura Lascasiana

The Int and Xis proteins are derived from the TnP7d encoded genes, int and xis, located at the right end o f the element (Figure 1.2). Int is required for excision and insertion and is a member o f the integrase family o f site-specific recombinases. It is related to integrase proteins from a variety o f different genomes including bacteriophage X (Flannagan et al.,

1994). Xis is required for excision (Senghas et a l, 1988; Poyart-Salmeron et a/., 1989; Jaworski et a l, 1996; Rudy et a l, 1997b) and is most closely related to an excisionase enzyme from the vancomycin resistance encoding transposon Tn5382 (Carias et a l,

1998).

Int contains two DNA binding domains (Lu and Churchward 1994). The C-terminal domain binds to both ends o f the transposon and protects a 40 bp region o f transposon and flanking DNA in a DNase protection assay (Lu and Churchward 1994). A 40 bp region is also protected in the target, centred on the insertion site. Circular permutation assays showed a static bend in the target sequence. Int mediates specific DNA cleavage at both the left and right ends o f TnP7d (Taylor and Churchward, 1997). Double stranded staggered cuts are made, which leave 5' single stranded protruding ends (Figure 1.3a). It is these ends which form the joint in the circular molecule.

DNase protection assays with Xis showed that it bound specifically to sites close to the ends o f TnP7(5 (Rudy et a l, 1997b). The binding site contains the 11 bp direct repeat sequence DR2 and is located in the same relative position to the binding sites for the N- terminal domain o f Int (Scott and Churchward, 1995). The results suggest that Xis is

involved in the formation o f nucieoprotein structures at the ends o f the element which help to align the ends so that excision and circularization can take place.

The DNA protection pattern exceeded the region which Xis was expected to protect (based on the size o f the protein), suggesting that a structure may be formed where the DNA is wrapped around Xis. A bent region o f DNA will be protected over a larger region than a comparable straight stretch. Another possible explanation is that Xis may bind to the DNA in the crook o f the static bend. Therefore Xis may facilitate the binding o f Int to its specific target site by bending the DNA or by the formation o f protein-protein or protein-DNA complexes, or a combination o f both. Recently, Wojciak et al. (1999) demonstrated the NM R structure o f the TnP7d integrase-DNA complex and showed that the DNA was bent approximately 35°. These experiments were performed in the absence o f Xis but the DNA substrate for these experiments was a 13-mer which included the binding site for Int, therefore it is still possible that Xis could facilitate the bending o f the DNA and that Xis is required for the correct alignment o f the DNA strands and

subsequent binding to the Int molecules.

Marra and Scott (1999) reported that Xis is needed for excision; in the absence o f Xis excision o f TnP7d from a chloramphenicol resistance gene was observed at less than 3.1

X 1 0 ' ^ ^ (beyond the limits o f detection in this experiment). When Xis was provided in

trans the normal levels o f excision were restored. When xis was over expressed with

normal expression o f int there was no increase in the excision frequency. This was also true when int was over expressed and xis was expressed normally. When both xis and int

This strongly supports the view that both xis and int are needed for excision and the relative concentration o f each is the rate limiting step in the excision reaction which precedes all natural transposition and conjugation events o f this element.

The excision and circularization o f the element and closure or repair o f the donor

molecule occurs at identical frequencies, strongly suggesting that a single reaction causes both events (Marra and Scott, 1999). Over expression o f both xis and int caused the circular molecule to be present in high enough quantities to be detected by a plasmid preparation, the excised molecules were however, lost from the cells suggesting that the molecules were not re-inserting into the host genome. One factor that might prevent insertion into the host DNA is an excess o f Xis as is the case in the 1 system. For X,

insertion requires the presence o f IntA. alone and is inhibited by the over expression o f Xis

X (Leffers and Gottesman, 1998). The XisÀ protein prevents the binding o f Int^ to a site required for integration (Moitoso de Vargras and Tandy, 1991). In E. coli only Int is required for integration o f TnP7d (Lu and Churchward, 1994). Using prim er extensions and RT-PCR the presence o f two transcripts containing int have been shown from the RNA o f B. subtilis. One o f these transcripts was initiated at a promoter located between

int and xis, the other transcript contained xis initiating presumably at a promoter upstream o f xis. This suggests that there are steps in conjugative transposition that require Int but not Xis, (Marra and Scott, 1999).

The various protection experiments can, together with a comparison o f the bacteriophage

interactions between Int, Xis and the arms of the transposon during excision o f TnPid, (Figure 1.4). The model, proposed by Scott and Churchward (1995), assumes that 4 Int molecules align the two DNA strands in an antiparallel arrangement, this is presumably catalysed by the presence o f Xis. One Int molecule bound to the right end o f TnPM at the T ’ site by its C-terminus and to N ’ 1 by its N-terminus forms an intrastrand loop. A second Int molecule binds the left end of the transposon at the T site by its C-terminus and the N ’2 site o f the right end o f the transposon by its N-terminus, forming a bridge between the left and right end o f the transposon. The third Int molecule binds to the B site o f the right end and the N2 site at the left end forming another bridge between the two transposon ends. The fourth Int molecule binds to the B ’ and N1 sites o f the left end forming another intrastrand loop. It may however be a truncated Int molecule possessing only the C-terminus, sites N1 and N2 have only a single corresponding site in phage X.

Therefore in the diagram the fourth Int molecule is shown bound only by its C-terminus.

The o r f encoding Int has other plausible start codons with ribosome binding sites in the

correct positions, and these could provide the truncated form o f Int that may function in this model. (Scott and Churchward, 1995)

A. B ’ C T DR2 N I N 2 N 3 DR2 A N ’2 N ’ I T ’ C ’ B B. N ’2 NI D N A B ending N2 N ' 2 N I N 2

Plan view Side view

F i g u r e 1.4. A. P o s it i o n o f X i s a n d Int b i n d i n g sites w ith in l n 9 1 6 . T h e th ic k b l a c k line r e p r e s e n t s T n P / 6 a n d

th e thin b l a c k line r e p r e s e n t s f l a n k i n g b a c te r ia l D N A . L a n d R r e p r e s e n t th e left a n d ri g h t e n d o f th e e l e m e n t

re s p e c t iv e l y . T h e o p e n t r ia n g l e s r e p r e s e n t th e r e g i o n s p r o t e c t e d b y Xis. T h e filled t r ia n g l e s l a b e l le d N l , N 2 ,

N 3 , N 1 ’ a n d N 2 ’ r e p r e s e n t th e r e g io n s b o u n d b y the N - t e r m i n a l d o m a i n o f Int. T h e filled d i a m o n d s l a b e lle d

B a n d T ’ a n d l a b e l le d B ’ a n d T r e p r e s e n t th e r e g i o n s b o u n d b y th e C - t e r m i n u s o f Int. T h e a r e a la b e l le d C

a n d C ’ r e p r e s e n t th e c o u p l i n g s e q u e n c e s at e a c h e n d o f th e e le m e n t . B. M o d e l f o r t h e a l i g n m e n t o f th e left

a n d rig h t e n d s o f T n P / 6 d u r i n g e x c isio n . T h e r ig h t a n d left p a r ts o f th e f i g u r e r e p r e s e n t t w o d i f f e r e n t v i e w s

o f the s a m e s t r u c tu r e . T h e g r e y line r e p r e s e n t s th e r ig h t e n d o f T n P / 6 a n d th e b l a c k line r e p r e s e n t s th e left

end. T h e p o s i t i o n s o f th e Int b i n d i n g si tes are in d ic a te d as is th e r e la tiv e p o s i t i o n s o f th e c o u p l i n g s e q u e n c e s

p r e s e n t at e i t h e r e n d o f th e t r a n s p o s o n . In th e left p a rt o f F ig u r e 1.7B th e larg e c ir c le s r e p r e s e n t th e C-

cylinders represent the C-terminal domain and the small circles represent the N-terminal domain. The hatched circle on the left and the hatched cylinder on the right represent an Int m olecule, which may have two D N A binding domains or only a C-terminal domain. The hatched shape labelled D N A bending represents a hypothetical host factor that binds and bends the D N A between T ’ and N ’ 1. (Adapted from Scott and Churchward, 1995; Rudy et a l , 1997b).