Emma Wampler '09
DNA transposition, a process whereby defined DNA
segments move freely about the genome, has extensively affected
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.
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.
domain contains approximately 300 residues, and contains the
"ribonuclease H-like motif."
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.
folding motif in Tn5 transposase
overlaps closely with the catalytic domains of
the retroviral integrases and Mu transposase.
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
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
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.
recognition helix is within hydrogen
bonding distance of oxygen 4 of thymine
10 of the
cis binding interactions
are provided by the amino-terminal end
of helix 2, which contains three arginine residues
that form salt
bridges with phosphates in the DNA backbone.
residues from the catalytic domain, Arg342, Glu344,
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.
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,
here is the asparagine and glutamate
coordinating with the 3' OH group of the transferred strand a maganese
is bound by Tn5 Transposase occasionally
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.
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.
D. R.; Goryshin, I. Y.; Reznikoff, W. S.; Rayment, I. 2000.
Three-Dimensional Structure of the Tn5 Synaptic Complex Transposition
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
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