Mechanism of DNA Replication 
Michael E. O'Donnell, Ph.D. — Assistant Investigator 
Dr. O'Donnell is also Associate Professor of Microbiology at Cornell University Medical College, New York 
City. He received his Ph.D. degree from the University of Michigan, Ann Arbor, where his research on 
electron transfer in the flavoprotein thioredoxin reductase was conducted with Charles H. Williams, Jr. 
He performed postdoctoral work on Escherichia coli replication with Arthur Kornberg and then on herpes 
simplex virus replication with Bob Lehman, both in the Biochemistry Department at Stanford University. 
MY laboratory is studying the duplication of 
genetic information. By understanding the 
fundamental mechanisms of cell growth, or the 
replication of DNA, we may obtain insights into 
the development of abnormal cells, including tu- 
mor cells. 
The genetic material of our cells, the chromo- 
somes, is a library with all the information 
needed for the multitude of duties required to 
maintain the cell's life. Included in these duties 
is the buildup of complete, new cellular machin- 
ery for the synthesis of another cell (reproduc- 
tion). The chromosome library is made of two 
long interwound helical fibers of DNA (deoxyri- 
bonucleic acid polymers). Before a cell can di- 
vide to form two new cells, it must duplicate the 
genetic library so that each cell has a complete 
copy of instructions on how to live. 
The process of duplicating DNA is intricate, 
and the cell has evolved a precision machine to 
carry out this important task. Its several protein 
parts are like gears of a machine, which coordi- 
nate their actions to unzip and unwind the dou- 
ble-helical strands of DNA. The machinery then 
uses the separated single strands as templates to 
synthesize two double-helical daughter chromo- 
somes. Subsequently these will segregate in two 
newly formed cells. 
Our goal is to understand, at a molecular level, 
the workings of proteins in the mechanics of DNA 
duplication. The system we are studying is the 
bacterium Escherichia coli, a relatively simple 
organism. The protein machine that duplicates 
the E. coli chromosome is called DNA polymer- 
ase III. The DNA polymerase III of E. co/i has nine 
accessory proteins plus the polymerase. Like the 
E. CO/? polymerase III, the DNA polymerases that 
replicate the chromosomes of higher organisms 
such as yeast, Drosophila, and humans are also 
composed of a DNA polymerase protein and sev- 
eral other "accessory" proteins. 
The function of the DNA polymerase protein 
(the a-subunit) is to synthesize the DNA. One of 
the accessory proteins, the e-subunit, is an exonu- 
clease that "proofreads" the product of the poly- 
merase protein. Very little is known about the 
functions of the other eight accessory proteins. 
However, since the several accessory proteins to 
the DNA polymerase are conserved in evolution 
from bacteria to humans, it seems reasonable to 
expect their individual functions to serve very 
important roles in the process of chromosome 
duplication. Analysis of the E. coli DNA polymer- 
ase III system will likely extend and generalize 
the understanding of the replication process in 
all organisms. 
We have recently developed methods to obtain 
pure preparations of each protein, or subunit, of 
the E. coli DNA polymerase III, and from these 
the whole complex can be reassembled. We have 
studied the individual subunits for biochemical 
activities and for their physical interactions. Two 
subunits, 7 and b, bind to each other to form a 
complex that, upon binding to primed DNA, hy- 
drolyzes ATP. In the presence of the /3-subunit, 
the 75 heterodimer couples the hydrolysis of ATP 
to clamp a dimer of the |8-subunit onto primed 
DNA. One molecule of the 76 heterodimer can 
clamp many (8-dimers onto primed DNA. Our bio- 
chemical studies indicated that the |8-dimer is 
clamped to DNA by encircling it like a doughnut. 
The x-ray structure of this /3-clamp has recently 
been solved in a collaboration with John Kuriyan 
(HHMI, Rockefeller University). It appears as a 
thin disk with a hole through the middle to ac- 
commodate the DNA. These studies on the /?- 
clamp and the 76 heterodimer were funded by 
the National Institutes of Health. 
The |8-clamp on DNA binds the polymerase sub- 
unit, tethering it to the DNA template. Whereas 
the polymerase alone is slow (20 nucleotides/ 
second), it is greatly accelerated upon binding 
the jS-clamp (700 nucleotides/second) and repli- 
cates an entire 8-kb single-strand circular DNA 
without coming off (processive). This fits nicely 
with the fact that the E. coli cell duplicates its 4 
million-base chromosome within 30 minutes. 
Another function of the 7-, 6-, and j8-subunits is 
to rapidly deliver the polymerase subunit from a 
completely replicated DNA molecule to a new 
primed DNA template. This rapid delivery of poly- 
merase is important because one strand of the 
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