Mechanism of DMA Replication 
Michael E. O'Donnell, Ph.D. — Assistant Investigator 
Dr. O'Donnell is also Assistant Professor of Microbiology at Cornell University Medical College, New York 
City. He received his Ph.D. degree with Charles H. Williams, Jr., from the Department of Biological Chem- 
istry at the University of Michigan, Ann Arbor, on electron transfer in the flavoprotein thioredoxin re- 
ductase. He performed postdoctoral work on Escherichia coii replication with Arthur Romberg 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, the chromosomes, 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 machinery for the syn- 
thesis of another cell (reproduction) . The chro- 
mosome library is made of two long interwound 
helical fibers of DNA (deoxyribonucleic acid 
polymers). Before a cell can divide 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. 
The aim of our research 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 E. coli chromo- 
some is replicated by over a dozen proteins. Ten 
of these are tightly bound into a complex that 
contains the DNA polymerase activity. Our pres- 
ent research is focused on this complex, called 
DNA polymerase III holoenzyme, which will be 
referred to below as "the holoenzyme." 
Only two proteins of the holoenzyme have 
well-defined functions: a, the DNA polymerase 
protein, and e, an exonuclease that proofreads the 
product of the polymerase protein. Our aim is to 
determine the individual functions of the other 
eight "accessory proteins" of the holoenzyme. 
We hope that analysis of the E. coli holoenzyme 
will extend and generalize the understanding of 
the replication process in all organisms. 
We have recently developed methods to obtain 
nearly pure preparations of each protein, or sub- 
unit, of the E. coli holoenzyme, 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 5, bind to each other to form a 
complex that, upon binding to primed DNA, hy- 
drolyses ATP. In the presence of the ^-subunit, 
the yb heterodimer couples the hydrolysis of ATP 
to clamp a dimer of the (S-subunit onto primed 
DNA. One molecule of the 7^ heterodimer can 
clamp many jS dimers onto primed DNAs. 
The /? 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 |S clamp (700 nucleotides/second) and repli- 
cates an entire 8-kb single-strand circular DNA 
without coming off (processive) . Hence three ac- 
cessory proteins of the holoenzyme (7, b, j3) are 
needed to confer rapid and processive synthesis 
onto the polymerase subunit. This fits nicely with 
the fact that the E. coli cell duplicates its 4 mil- 
lion-base chromosome within 30 minutes. 
After the holoenzyme has replicated the DNA, 
it remains bound to it. However, upon addition of 
primed DNA containing a |8 clamp, the new clamp 
specifically seeks out polymerase molecules 
bound to completed DNA templates, and the /? 
clamp on the fresh primed DNA "steals" the poly- 
merase away from the clamp on the completed 
template. Hence the 13 protein appears to acceler- 
ate the polymerase as well as mediate its rapid 
cycling from a completed DNA to a new one. The 
rapid cycling is important because one strand of 
the DNA duplex (lagging strand), as a result of 
the geometry of the DNA helix, must be repli- 
cated in fragments. Synthesis of these fragments 
requires that the polymerase be used over and 
over (cycling) every 1-2 seconds. 
Two T-subunits bind tightly to each other (di- 
meric), and each binds a polymerase molecule. 
Hence the r-subunit dimer serves as a scaffold to 
331 
