Howard Hughes Medical Institute
Department of Molecular Biology and Genetics
Johns Hopkins School of Medicine
725 North Wolfe Street, Baltimore, Maryland 21205
To contact Nancy: ncraig@jhmi.edu To contact the lab: 410. 955.2731
410. 955.3933
Nancy is a member of the Biochemistry, Cellular and Molecular Biology (BCMB) Graduate Program
TnsA + TnsB form the transposase which recognizes specific sequences at the ends of the transposon and executes the DNA breakage and joining reactions that underlie transposition. TnsC is a regulator of transposition that interacts with TnsAB to promote transposition when TnsC is in contact with either of the target-determining proteins TnsD or TnsE and an appropriate target DNA. TnsD directs Tn7 insertion into attTn7 whereas TnsE directs insertion into many different sites unrelated to attTn7, Interestingly, the TnsE pathway preferentially directs insertion into bacterial plasmids that can move between cells, thus promoting the spreading of Tn7 from organism to organism, a process important in the rapid dissemination of antibiotic resistance among bacterial populations.
Our overall goals are to understand in molecular detail how Tn7 moves from place to place and how the frequency of this movement is modulated. We expect that understanding the macromolecular interactions that underlie Tn7 transposition will contribute not only to the understanding of DNA recombination but also to the understanding of other complex protein–nucleic acid transactions, such as DNA replication, transcription, and RNA processing.
The fundamental steps in transposition are the DNA cleavages that separate the transposon from its flanking donor DNA and the subsequent joining of the transposon ends to the target DNA. We are dissecting the mechanism of these DNA strand breakage and joining reactions during Tn7 transposition. Our strategies include analyzing recombination under altered reaction conditions, using DNA substrates derivatized in particular ways, and using genetic approaches to isolate and characterize mutant proteins altered in their ability to promote these reactions.
At the heart of the Tn7 transposition machinery is a higher-order protein-DNA complex in which the transposon ends are apposed to the target DNA by TnsA and TnsB. These two Tns proteins collaborate to execute the breakage and joining reactions that underlie transposition and thus together form the Tn7 transposase, TnsAB. The other Tns proteins are regulatory proteins that define when and at what target sites recombination will occur. TnsB also binds specifically to the ends of the transposon, thereby identifying these positions as recombination sites; TnsA is likely recruited to the Tn7 ends through interactions with TnsB. Both TnsA and TnsB play direct roles in the breakage and joining reactions: each of these proteins contains an active site for a DNA-processing reaction. Interestingly, the active sites of TnsB proteins appear to be fundamentally related to the active sites of other recombinases, including retroviral integrases, whereas the active site of TnsA is fundamentally related to those of restriction enzymes.
The transposase TnsAB is not constitutively active; TnsAB activity is modulated and directed to particular target sites by the other Tns proteins. We have isolated gain-of-function derivatives of both TnsA and TnsB that result in transposase activity in the absence of the other Tns proteins. Understanding the basis of the activation of these proteins will provide insights into how recombination by wild-type Tns proteins is modulated. Another strategy we are using to dissect TnsA and TnsB is to isolate sufficient quantities of these proteins for structural analysis. We are also making other mutant derivatives of these proteins to examine their interactions with each other and with DNA. In a collaboration with colleagues at the NIH, a cocrystal of TnsA with part of the TnsC regulator has been obtained, allowing us to probe at very high resolution the nature of the interactions between these proteins.
TnsC plays a central regulatory role in Tn7 transposition: TnsC interacts with both TnsA and TnsB to activate the transposase activity and also plays a key role in target site selection by interacting with the target DNA and a targeting protein, TnsD (and likely TnsE and TnsE target DNA). We have been able to identify protein-DNA complexes containing the donor DNA and the target DNA and the Tns proteins. Formation of this complex is a key intermediate and regulatory step in transposition.
Tn7 transposition is directed to attTn7 by the specific binding of TnsD to attTn7 and the formation of a TnsC-TnsD-attTn7 target complex. TnsD interaction with attTn7 generates an altered DNA structure that is recognized by TnsC. Because the altered target DNA is part of the DNA insertion site, the breakage and joining reactions actually occur on DNA molecules, not simply in solution, as could occur if these reactions were prompted only via protein-protein interactions. However, we have also found that protein-protein interactions between TnsC and TnsD play an important role in transposition.
We are also investigating Tn7 insertion into sites other than E. coli attTn7. Related sites are present in virtually all organisms because the binding site for TnsD actually lies within a highly conserved gene. We have investigated the target activity of the human attTn7 sequence and found naked DNA containing this segment is a good target for Tn7. However, we have been unable to observe Tn7 insertion into attTn7 DNA that is bound by nucleosomes, a finding that may well preclude using Tn7 for targeting insertions in mammalian cells, a process that could be useful in gene therapy.
We are also using Tn7 to explore the structure of the E. coli chromosome by making large libraries of Tn7 chromosomal insertions and then analyzing their distribution using microarray methods. We have found very interesting global patterns of insertion, for example insertion around the region of the origin of replication is particularly high. Using this methodology, we have also found that sites that are widely separated, i.e. by many 100s of kbp between sites on the linear chromosome, can strongly influence each other.