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Tn5 Transposase

Emma Wampler '09


I. Introduction

DNA transposition, a process whereby defined DNA segments move freely about the genome, has extensively affected evolution. Transposition is mediated by proteins called transposases,which bind to two strands of DNA, cut one, and reattach it to the other. This introduces new genomic material into the host genome. Click here for a cartoon of how transposase works5.

The first such processes discovered in eukaryotes was by Barbara McClintock, in maize4. Retroviruses such as human immunodeficiency virus-I catabolize similar processes by which they reproduce their genome using the host's cells3.

Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition. This structure provides a molecular framework for understanding many aspects of transposition1. Understanding how transposases interact with DNA could serve to help develop anti-retrovirus drugs2.

The transposon end DNA is bound by one subunit of the transposase, another catalyzes cleavage, and a third active site, a hairpin intermediate of the protein, cleaves the two strands of DNA and transfers the strands into target DNA1.  

II. General Structure

Tn5 Transposase is a dimer of two chemically identical 476-residue monomers. The overall structure of the monomer is assymetric and can be described by three major domains. 

The 70-residue N-terminal domain consists entirely of alpha helices and turns and functions primarily to bind DNA.

The central catalytic domain contains approximately 300 residues, and contains the "ribonuclease H-like motif." This structural motif is an alpha/beta/alpha fold with a mixed beta sheet of five strands that has strand 2 antiparallel to the other four strands and contains all the amino acid residues that make up the catalytic active site. This folding motif in Tn5 transposase overlaps closely with the catalytic domains of the retroviral integrases and Mu transposase. 

The COOH-terminal contains around 100 residues.  

III. DNA Binding

The first 17 bp of the sequence engage in protein-DNA contacts, with 12 of these base pairs engaging in sequence-specific protein-DNA contacts. DNA binding elements for Tn5 transposase are widely spread over nearly the entire primary sequence of the transposase protein. Both dimers interact with each DNA molecule.

DNA bound to transposase differs from standard B-DNA conformation. When Tn5-Transposase binds to DNA, it bends the DNA.

The 70 residues of the amino-terminal end of Tn5 transposase form a domain that binds DNA in cis. This domain is formed from four a-helical segments, instead of the three found in the HTH domains. The fourth a-helix in Tn5 acts as the major "recognition helix" and makes several base pair-specific contacts in the major groove of the DNA from positions 7 to 13. Residue Lys54 of the recognition helix is within hydrogen bonding distance of oxygen 4 of thymine 10 of the transferred strand. Other cis binding interactions are provided by the amino-terminal end of helix 2, which contains three arginine residues (Arg26, Arg27, and Arg30) that form salt bridges with phosphates in the DNA backbone. Three residues from the catalytic domain, Arg342, Glu344, and Asn348 also contact DNA in cis.

Many of the trans protein-DNA interactions are provided by the interaction of a segment of anti-parallel beta-sheet that sticks out from the catalytic domain between residues Ile239 and Lys260. This secondary structural motif clamps down on the DNA close to the active site via major groove binding. Other significant trans protein- DNA contacts occur near the active site.

IV. The Catalytic Domain

A catylitic triad of acidic residues creates the DDE motif, which form a binding site for magnesium that is required for catalysis. These three acidic residues are Asp97, Asp 188, and Glu326. Shown here is the asparagine and glutamate coordinating with the 3' OH group of the transferred strand a maganese atom. Maganese is bound by Tn5 Transposase occasionally instead of magnesium, and this binding affinity is increased in some mutant forms of the protein.

The maganese primes the 3' OH end of the transferred strand for nucleophillic attack on the target DNA. This creates an intermediate hairpin structure. Several DNA-protein interactions in the active site help the 3' OH group be placed close enough to the 5' end of the target strand for this nucleophillic attack by stabilizing a bend in the backbone of the target strand. A thymine second to the end of the 5' end is flipped out of the DNA helix and held in a hydrophobic bindinb pocket so that it is able to form stacking interactions with Trp298.

V. Practical Applications

Because the structure and sequence of this tranposase seems to be highly similar to that of retroviruses, specifically HIV, knowledge of how this tranposon binds to and cleaves DNA could be of use to develop new drugs to fight HIV. Specifically, Lys330 and Lys333 are analogous to Lys156 and Lys159 of HIV-1.Click here for a cartoon of the similarities between Tn5 transposase and HIV-1 integrase6. Recent drug leads suggest that a synaptic complex of HIV-1 integrase may be a promising drug target2. Because of the similarity of the catalytic active sites and overall catalytic core structure of all transposaselintegrase enzymes, the Tn5 transposase synaptic complex structure may be an important first step toward modeling the integrase-DNA interaction of HIV-1 integrase, and provide the basis for potential antiviral therapies for the treatment of AIDS.

VI. References

1Davies, D. R.; Goryshin, I. Y.; Reznikoff, W. S.; Rayment, I. 2000. Three-Dimensional Structure of the Tn5 Synaptic Complex Transposition Intermediate. Science 289:77-85.

2Hazuda, D. J.; Felock, P.; Witmer, M.; Wolfe, A.; Stillmock, K.; Grobler, J. A.; Espeseth, A.; Gabryelski, L.; Schleif, W.; Blau, C.; Miller, M. D. 2000. Inhibitors of Strand Transfer That Prevent Integration and Inhibit HIV-1 Replication in Cells. Science 287:646-650.

3Yang, Zhong-Ning; Mueser, Timothy C.; Bushman, Frederic D.; Hyde, C. Craig. 2000. Crystal Structure of an Active Two-domain Derivative of Rous sarcoma virus integrase. J. of Mol. Bio. 296:535-548.

4McClintock, Barbara. 1950. The Origin and Behavior of Mutable Loci in Maize. Proceedings of the National Academy of Sciences of the United States of America 36:344-355.



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