mediate low-frequency insertion into many differ- 
ent random sites. 
Transposition involves specific recognition of the 
transposon ends and the target DNAs, juxtaposition 
of these substrates, strand cleavage to excise the 
transposon and expose its termini, and strand 
transfer to join the 3'OH transposon ends to the tar- 
get. These breakage and joining reactions appear to 
occur in an elaborate protein-DNA complex con- 
taining the substrate DNAs and the Tns proteins. 
Roles for some of the proteins have been estab- 
lished. TnsB plays a central role in transposon recog- 
nition, because it is a specific DNA-binding protein 
that interacts with both Tn7 ends. A non-sequence- 
specific DNA-binding protein whose interaction 
with DNA requires ATP, TnsC plays a key role in 
target recognition. The role of TnsA remains to be 
established clearly; one attractive hypothesis is that 
TnsA mediates interaction (s) between TnsB, bound 
to the transposon ends, and TnsC, bound to the tar- 
get DNA. How do TnsD and TnsE activate the core 
machinery? TnsD is a sequence-specific DNA- 
binding protein that binds specifically to attTnl 
and promotes the interaction of TnsC with attTnl, 
thereby bringing the core machinery and transposon 
ends to the insertion point. Perhaps TnsE is a low- 
specificity DNA-binding protein that positions the 
core machinery at many places on target DNA. 
Dissecting the Mechanism of DNA Strand 
Breakage and Joining 
The fundamental reactions in transposition are 
DNA strand cleavage to expose the transposon ends 
and strand transfer to join the ends to the target DNA. 
The current major focus of the research in Dr. 
Craig's laboratory is to determine in chemical detail 
how these reactions occur and to define the active 
sites that promote them. 
It is suspected that these activities are provided by 
TnsB, the protein that interacts specifically with the 
transposon ends. This hypothesis arose because Dr. 
Craig and her colleagues were able to align part of 
the TnsB amino acid sequence with highly con- 
served sequences of several retroviral and retrotrans- 
poson integrases; others have hypothesized that this 
conserved motif plays a key role in DNA strand cleav- 
age and transfer by providing an essential metal co- 
factor-binding site. 
Several experimental approaches are being used 
to determine if TnsB does indeed contain the active 
site for transposition. One strategy will be to change 
highly conserved amino acids in TnsB by site- 
directed mutagenesis. A more powerful approach is 
to isolate gain-of-function TnsB mutants in which 
these usually repressed activities are artificially acti- 
vated. Dr. Craig and her colleagues are seeking such 
mutants in the presence of various combinations of 
the other Tns proteins because they suspect that 
multiple protein-protein interactions and protein- 
DNA interactions modulate the activity of TnsB. 
Another strategy is to use alternative or modified 
DNA substrates. Using work with retroviral inte- 
grases as a base, Dr. Craig and her colleagues are 
developing a novel Tn7 substrate in which a tran- 
sposon end is already joined to one strand of the 
target DNA. Others have shown that the exposed 
target end can participate in novel transposase- 
dependent reactions of reduced stringency, provid- 
ing a powerful tool for examining low-level uncou- 
pled reactions and the strand transfer chemistry. 
This novel strand transfer reaction with the Tns pro- 
teins has been observed in this laboratory, and the 
requirements and characteristics of this unusual re- 
action are being examined. 
Dr. Craig and her colleagues also wish to examine 
in detail the elaborate protein-DNA complex in 
which transposition occurs and, in particular, to de- 
fine the specific interactions between the various 
Tns proteins. They anticipate that affinity chroma- 
tography and protein crosslinking will be important 
tools in this work. 
Control of Tn7 Transposition 
Tn7 transposition is highly controlled. For exam- 
ple, although TnsA + TnsB + TnsC contains the ac- 
tive sites for transposition, no transposition occurs 
unless the activators TnsD or TnsE are also present. 
The interaction of TnsC with the target DNA is a 
critical control step. For example, changing TnsC's 
ATP cofactor has a profound effect on transposition; 
moreover, TnsD appears to activate transposition by 
promoting the stable interaction of TnsC with 
attTn 7. TnsC mutants that activate the core machin- 
ery in the absence of TnsD or TnsE have recently 
been isolated. A number of amino acid changes that 
provide activated TnsC have been identified. Under- 
standing why these mutant proteins are activated 
will provide further insight into the control of wild- 
type TnsC. Another complementary approach is the 
effort in Dr. Craig's laboratory to reconstitute TnsE- 
dependent transposition in vitro with purified pro- 
teins. Being able to manipulate both the high- 
frequency TnsD pathway and low-frequency TnsE 
pathway in vitro will be useful in dissecting transpo- 
sition control. 
Another feature of Tn7 transposition that reflects 
the highly controlled nature of this reaction is that 
Tn7 displays transposition immunity; that is, Tn7 
40 
