Structural Studies on DNA-Replication 
Enzymes, src- related Oncogene Products, 
and Oxidoreductases 
John Kuriyan, Ph.D. — Assistant Investigator 
Dr. Kuriyan is also Associate Professor of Molecular Biophysics at the Rockefeller University. He received a 
B.S. degree in chemistry from Juniata College, Pennsylvania, and a Ph.D. degree from the Massachusetts 
Institute of Technology, where he worked with Gregory Petsko and Martin Karplus on the dynamics of 
proteins. He continued with Martin Karplus at Harvard University for a year and then moved to the 
Rockefeller University as a University Fellow. He is also a Pew Scholar in the Biomedical Sciences. 
OUR interests are in understanding the struc- 
tural determinants of protein function. To 
this end we carry out x-ray diffraction experi- 
ments and computer simulations aimed at charac- 
terizing the three-dimensional structures of pro- 
teins. We apply the knowledge gained from these 
studies toward the design of mutations and inhibi- 
tors to modify protein activity, and seek, from the 
particular cases in hand, to extrapolate to fami- 
lies of related proteins. Our current efforts are 
focused on three main areas: DNA replication, 
5rc-related oncogene products (tyrosine ki- 
nases), and redox enzymes and oxidative stress. 
Structure of the /S-Subunit 
of DNA Polymerase III 
DNA polymerases are enzymes that duplicate 
the information content of DNA by catalyzing the 
template-directed polymerization of nucleic 
acids. Polymerases that are involved in chromo- 
somal replication, such as DNA polymerase III 
(PolIII) of Escherichia colt, are distinguished by 
their high processivity — i.e., their rapid replica- 
tion (1 ,000 bases/second) of very long stretches 
of DNA without dissociation. Processivity is con- 
ferred upon the enzyme by one of its subunits, /3 
(processivity factor), which acts to clamp the 
polymerase onto DNA. The /3-subunit binds very 
strongly — indeed, cannot be easily separated 
from circular DNA. It has been shown, however, 
to slide freely along duplex DNA, consistent with 
its role as a clamp that tethers the polymerase 
core to the template and advances with the poly- 
merase during replication. 
We have crystallized the |S-subunit and deter- 
mined its structure by x-ray diffraction to 2.5-A 
resolution (Xiang- Peng Kong, Rockefeller Univer- 
sity; Michael O'Donnell [HHMI] and Rene Onrust, 
Cornell University Medical College) . Two mole- 
cules of the /3-subunit are tightly associated to 
form a donut-shaped structure that forms a closed 
ring around duplex DNA. An unexpected feature 
of the structure is that it is highly symmetric. 
Each monomer consists of three domains of iden- 
tical chain topology. Each domain is roughly 
twofold symmetric in its architecture, with an 
outer layer of two (8-sheets providing a scaffold 
that supports two a-helices. Replication of this 
motif around a circle results in a rigid molecule 
with 1 2 a-helices lining the inner surface of the 
ring and with six jS-sheets forming the outer 
surface. 
The high symmetry of the structure is well 
suited to interact with the cylindrically symmet- 
ric DNA duplex, and the hole in the middle of the 
ring is large enough to accommodate either A or B 
forms of DNA with no steric repulsion. Although 
the overall charge on the protein is negative, a 
number of basic side chains on the inner surface 
generate a positive electrostatic field localized in 
the hole of the donut, precisely where the nega- 
tively charged phosphate backbone of DNA is ex- 
pected to be. We are carrying out molecular dy- 
namics simulations of a duplex-DNA and 
(8-subunit complex, which is expected to provide 
information not readily accessible by x-ray crys- 
tallography. Other extensions of this project in- 
clude crystallization attempts on the eukaryotic 
processivity factor PCNA (proliferating cell nu- 
clear antigen) , other subunits of PolIII, and other 
proteins acting at the replication fork. 
Oncogene Products Related to src 
Tyrosine kinases such as the product of the src 
gene play a central role in signal transduction 
pathways in the cell, and abnormalities in their 
function can result in dramatic changes in cellu- 
lar differentiation and life cycles. Our approach 
is to work on these proteins in isolation, and we 
are collaborating with Hidesaburo Hanafusa 
(Rockefeller University) and Marilyn Resh 
(Sloan-Kettering Institute) to crystallize various 
functional domains of these oncogene products 
and their cellular equivalents. Initial success has 
been obtained for the src SH2 domain, which is 
responsible for binding to proteins containing 
phosphorylated tyrosine residues. Starting from 
src-containing plasmids (provided by Resh), 
Dorothea Kominos and Gabriel Waksman in our 
laboratory have cloned, overexpressed, purified, 
and crystallized SH2. The crystals diffract to 2.5-A 
resolution, and the x-ray structure determination 
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