Structural Studies of Protein-Nucleic Acid Interaction 
smaller domain has the active site for the exonu- 
clease activity. Using site-directed mutagenesis, 
we have made an enzyme devoid of the editing 
exonuclease activity and determined its struc- 
ture. We have grown two crystal forms of this 
protein complexed with a small DNA substrate. A 
high-resolution structure of one crystal form 
shows a single-stranded tetranucleotide bound to 
the exonuclease active site. The second crystal 
form, grown under conditions when the enzyme 
is active, is presumed to have DNA at the polymer- 
ase active site and has recently been solved. Al- 
though there are clear changes in the structure of 
the polymerase domain, no bound duplex DNA 
has yet been visualized. These structures begin to 
address the issues of how these two active sites 
work together on the same DNA substrate and 
how they both function to enhance the DNA- 
copying fidelity of this and other polymerases. 
Genetic Recombination 
We have recently determined the crystal struc- 
tures of two proteins that are involved in genetic 
recombination. One protein, called resolvase, 
catalyzes a site-specific recombination between 
two duplex DNAs of identical sequence. Resol- 
vase is the product of a transposable element (a 
jumping gene) that can move throughout the E. 
colt population spreading drug resistance genes. 
This protein can bind to a specific duplex DNA 
sequence, align two DNA segments having the 
same sequence, cleave the two DNA duplexes, 
rearrange the duplexes, and re-ligate them, re- 
sulting in a recombinational event. We have de- 
termined the structure of the catalytic domain of 
this enzyme at 2.5 A resolution. This structure 
helps to explain the phenotypes of many resol- 
vase mutant proteins but does not suggest a mech- 
anism for this recombinase. We have recently co- 
crystallized this protein with a 31 -bp fragment 
that contains the recombination site, whose 
structure should provide clues to the mechanism 
of this reaction. 
E. colt recA protein is essential for general re- 
combination in E. colt. Using the energy of ATP 
hydrolysis, recA protein promotes the pairing of 
homologous duplex DNAs in preparation for re- 
combination. The structure of recA protein has 
been refined at 2.3 A resolution. The subunit 
forms a helical filament in the crystal very similar 
to that formed on DNA and thus enables us to 
understand the many mutant recA proteins made 
during the past decade and relate its structure to 
its functions in nucleotide binding, DNA binding, 
and the SOS response. Our goal is to understand 
how ATP hydrolysis and the homologous pairing 
of DNA are coupled. 
mv Proteins 
We have made large quantities of two proteins, 
reverse transcriptase and fragments of an activa- 
tor protein called Tat, that are specified by HIV 
(human immunodeficiency virus). Toward our 
goal of crystallizing and establishing the struc- 
tures of biologically relevant complexes, we have 
shown that fragments of Tat bind specifically to a 
portion of the HIV RNA called TAR. Initiation 
complexes formed using human tRNA'''^, an HIV 
RNA fragment, and reverse transcriptase are being 
explored. We have recently calculated an excel- 
lent 5 A resolution electron density map of re- 
verse transcriptase and should soon extend this 
study to 3-5 A resolution. Our long-term goal is 
to provide a structural basis for designing inhibi- 
tors of these proteins that might function as use- 
ful anti-AIDS drugs. 
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