David B. Haniford, PhD

 

 

Research Area:
The research in our lab is focused on mechanisms of DNA transposition and factors that regulate DNA transposition. In bacteria, transposons often acquire antibiotic resistance genes. The mobilization of these genes along with the transposon DNA is a major pathway for the spread of antibiotic resistance genes. A better understanding of how transposons are mobilized and the factors that regulate their movement is important for preventing the widespread dissemination of antibiotic resistance genes to pathogenic bacteria. We are currently using two related models systems, Tn10 and Tn5, to study transposition mechanisms and their regulation.

 

Regulation of Tn10 transposition by H-NS
Under certain growth conditions Tn10 transposition can be almost completely dependent on the nucleoid-associated protein, H-NS. Interestingly, H-NS is a central player in stress response and virulence pathways in gram-negative bacteria. This raises the possibility that, through regulation by H-NS, Tn10 transposition may be responsive to changes in environmental conditions. Such a signal transduction network could help ensure the continued propagation of Tn10 by activating transposition when growth conditions become suboptimal. We are currently working towards understanding how H-NS regulates Tn10 transposition and how H-NS activity is itself regulated in the cell. Our initial results from in vitro analysis of the Tn10 transposition reaction indicate that H-NS impacts the reaction by directly interacting with transposition intermediates, a novel role for this important regulatory protein. H-NS function is regulated by heterodimerization with a host of other proteins and we are very interested in determining if such interactions provide a means of regulating Tn10 transposition in response to a variety of different growth conditions.

Structural studies on Tn5 and Tn10 transpososomes
For DNA-based transposons the chemical steps in transposition take place in the context of a higher order protein-DNA complex called the transpososome. The three dimensional structure of the Tn5 transpososome was determined by X-ray crystallography. This structure provided valuable insights into the protein-DNA interactions that govern transpososome formation and steps in transposon excision. Subsequent to transposon excision the transpososome interacts with a target site and directs the joining of the transposon ends to the target DNA. Very little is known of the molecular interactions that direct target binding and insertion in transposition systems. We have established conditions for Tn5 transpososome binding to a single target site and would like to pursue X-ray crystallography of this new Tn5 transpososome. We are also working towards defining the structure of Tn10 transpososomes. We anticipate that this will provide unique insights into how target site selection occurs and how the strand joining events are catalyzed.

Representative Publications:

Kennedy, A.K., Guhathakurta, A., Kleckner, N. and Haniford, D.B. (1998). Tn10 transposition via a DNA hairpin intermediate. Cell 95, 125-134.

Allingham, J.S., Pribil, P.A. and Haniford, D.B. (1999). All three residues of the Tn10 transposase DDE catalytic triad function in divalent metal ion binding. Journal of Molecular Biology 289, 1195-1206.

Santagata, S., Besmer, E., Villa, A., Bozzi, F., Allingham, J.S., Sobacchi, C., Haniford, D.B., Vezzoni, P., Nussensweig, M.C., Pan, Z.-Q. and Cortes, P. The RAG1/RAG2 complex constitutes a 3’ flap endonuclease: implications for junctional diversity in V(D)J and transpositional recombination. Molecular Cell 4, 935-947.

Kennedy, A.K., Haniford, D.B., and Mizuuchi, K. (2000). Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: Insights from phosphorothioate stereoselectivity. Cell 101, 295-305.

Pribil, P.A. and Haniford, D.B. (2000). Substrate recognition and induced DNA deformation by transposase at the target-capture stage of Tn10 transposition. J. Mol. Biol. 303, 145-159.

Allingham, J.S., Wardle, S.W. and Haniford, D.B. (2001). Determinants for hairpin formation in Tn10 transposition. EMBO Journal, 20, 2931-2942.

Allingham, J.S. and Haniford, D.B. (2002). Mechanisms of metal ion action in Tn10 transposition. J. Mol. Biol. 319, 53-65.

Stewart, B.J., Wardle, S.J. and Haniford, D.B. (2002). IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration. EMBO J. 21, 4380-4390.

Pribil, P.A. and Haniford, D.B. (2003). Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition. J. Mol. Biol. 330, 247-259.

Swingle, B., O’Carroll, M., Haniford, D.B. and Derbyshire, K.M. (2004). The effect of host-encoded nucleoid proteins on transposition: H-NS influences targeting of both IS903 and Tn10. Mol. Micro. 52, 1055-1067.

Pribil, P.A., Wardle, S.J. and Haniford, D.B. (2004). Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA. Mol. Micro. 52, 1173-1186.

Humayun, S., Wardle, S.J., Shilton, B.H., Pribil, P.A., Liburd, J. and Haniford, D.B. (2005). Tn10 transposase mutants with altered transpososome unfolding properties are defective in hairpin formation. J. Mol. Biol. 346, 703-716.

Wardle, S.J., O'Carroll, M., Derbyshire, K.M. and Haniford, D.B. (2005) The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition. Genes and Development 19, 2224-2235.

Whitfield, C.R., Wardle, S.J. and Haniford, D.B. (2006). Formation, characterization and partial purification of a Tn5 strand transfer complex. J. Mol. Biol. 364, 290-301.

Ward, C.M., Wardle, S.J., Singh, R.K. and Haniford, D.B. (2007). The global regulator H-NS binds two distinct classes of sites within the Tn10 transpososome to promote transposition. Mol. Micro. 64, 1000-1013.

Singh, R.K., Liburd, J., Wardle, S.J. and Haniford, D.B. (2007). The nucleoid binding protein H-NS acts as an anti-channeling factor to favor intermolecular Tn10 transposition and dissemination. J. Mol. Biol. 376, 950-962.

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