The Mechanism of a Bacterial Transposition Reaction 
HHMI-supported effort is focused on dissecting 
the mechanism of the DNA strand breakage and 
joining reactions during Tn7 transposition. 
We previously developed a cell-free system for 
Tn7 transposition to attTnl. Tn7 inserts effi- 
ciently and specifically into attTnl in vitro in 
the presence of four purified TnV-encoded pro- 
teins: TnsA + TnsB + TnsC + TnsD. Thus these 
proteins participate directly in transposition. We 
have also established that Tn7 strand breakage 
and joining can be carried out by a subset of these 
proteins: TnsA + TnsB + TnsC. We now want to 
know which transposition protein carries out 
which particular step(s) in transposition and the 
chemical basis of these reactions. We are probing 
these questions by examining the ability of each 
transposition protein to individually execute a 
subset of the chemical steps that underlie the 
complete transposition reaction. Our strategies 
to reveal what may be usually suppressed activi- 
ties include manipulating the reaction conditions 
and using DNA substrates derivatized in particu- 
lar ways that we suspect may provoke strand 
cleavage and joining. 
Another biochemical method we are using to 
identify the domains of the recombination pro- 
teins that are most intimately involved in DNA 
strand breakage and joining is to determine, 
through protein-DNA crosslinking studies, the 
segments of the recombination proteins that most 
closely appose the positions of DNA strand break- 
age and joining during transposition. 
In a complementary genetic approach, we are 
seeking to identify "active sites" in the recombi- 
nation proteins that promote DNA strand break- 
age and joining, by isolating and characterizing 
mutant transposition proteins altered in their abil- 
ity to promote these reactions. We suspect that 
these active sites actually lie in TnsB, which binds 
specifically to the ends of Tn7, i.e., the sites of 
strand breakage and joining during transposition. 
We are also particularly interested in TnsB be- 
cause there is some sequence similarity between 
TnsB and the recombinases of retrotransposons, 
including the integrases of retroviruses. Our 
long-term goals are to describe in detail the chem- 
ical steps that underlie DNA breakage and joining 
and to understand how the recombination pro- 
teins promote these reactions. 
We are also interested in understanding how 
transposition is controlled and, in particular, in 
understanding the interplay between Tn7 and its 
bacterial host. 
Much of our biochemical characterization of 
Tn7 transposition has focused on dissecting the 
high-frequency insertion of Tn7 into its spe- 
cific insertion site attTn 7. We are now working 
to extend the biochemical analysis of Tn7 trans- 
position to its low-frequency insertion into ran- 
dom target sites. We are developing a cell-free 
system for Tn7 insertion into random target 
sites. We will then be poised to dissect the dif- 
ferences between high- and low-frequency Tn7 
transposition in molecular detail. We also sus- 
pect that characterization of Tn7 insertion into 
random target sites will reveal host proteins 
that likely participate in this recombination re- 
action. We are also examining how the struc- 
ture of the E. coli chromosome may influence 
Tn7 transposition. Another strategy we are us- 
ing to probe the control of Tn7 transposition is 
to isolate and characterize mutant Tns proteins 
that display altered transposition properties. 
The above work on the control of Tn7 transposi- 
tion is supported by a grant from the National 
Institutes of Health. 
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