Structural Studies of Protein-Nucleic Acid Interactions 
Replication of DNA 
E. co// DNA polymerase I functions primarily in 
the repair of DNA but is homologous to polymer- 
ases involved in replication. We have determined 
the structure of the Klenow fragment, a portion 
of Pol I that retains the polymerase and a 3'- to 
5'-editing exonuclease activity. We have shown 
that a larger structural domain, which has a cleft 
sufficient in size to bind duplex DNA, contains 
the active site for the polymerase reaction, 
whereas a smaller domain has the active site for 
the exonuclease activity. Using site-directed mu- 
tagenesis, we have made an enzyme devoid of the 
editing exonuclease activity and determined its 
structure. We have grown two crystal forms of 
this protein complexed with a small DNA sub- 
strate. A high-resolution structure of one crystal 
form shows a single-stranded tetranucleotide 
bound to the exonuclease active site and 1 1 base 
pairs of duplex DNA bound to a cleft that runs at 
right angles to the major cleft. There are changes 
in the structure of the polymerase domain, in- 
cluding the movement of a thumb-like structure. 
To access the polymerase active site in the cleft, it 
appears that the DNA will have to bend by 90°. 
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. This work is supported by a 
grant from the American Cancer Society. 
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. Resolv- 
ase is the product of a transposable element (a 
jumping gene) that can move throughout the E. 
coli 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 resolv- 
ase mutant proteins. This structure and that of the 
intact protein determined at 3-A resolution pro- 
vide ideas for a possible recombination mecha- 
nism. We have recently cocrystallized this pro- 
tein with a 31 -bp fragment containing the 
recombination site, whose structure should pro- 
vide further clues to the mechanism of this 
reaction. 
E. coli recK protein is essential for general re- 
combination in E. coli. Using the energy of ATP 
hydrolysis, recK protein promotes the pairing of 
homologous duplex DNAs in preparation for re- 
combination. The structure of rech. 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 recK 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. We have now produced 
good crystals of the next enzyme in the pathway 
of recombination, ruvC, which cleaves the DNA 
recombination intermediate called the Holliday 
structure. The work on resolvase and the recK 
protein is supported by a grant from the National 
Institutes of Health. 
HIV Proteins 
During the past year we have determined the 
structure of human immunodeficiency virus 
(HIV) reverse transcriptase complexed with a 
nonnucleotide inhibitor that shows promise as an 
anti-AIDS drug. The enzyme is a heterodimer of a 
66-kDa polypeptide containing a polymerase and 
RNase H domain and a 5 1 -kDa polypeptide con- 
taining only the polymerase domain. This di- 
meric polymerase shows an asymmetric structure 
with one active-site cleft running from the p66 
domain to the RNase H domain. The p5 1 subunit 
has no cleft. The similarities between the cata- 
lytic domains of HIV reverse transcriptase and 
Klenow fragment suggest that all polymerases 
have the same catalytic site. We have been suc- 
cessful in diffusing other nonnucleotide inhibi- 
tors into the crystal and find that they bind into a 
deep pocket near to, but not overlapping with, 
the polymerase catalytic site. We expect that de- 
sign of additional inhibitors based on the struc- 
ture will be possible. 
Recently a peptide fragment of the transactiva- 
tor protein Tat has been cocrystallized with a 
fragment of the RNA site to which it binds, TAR. 
This work is supported in part by a grant from the 
National Institutes of Health. 
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