Mechanism of DNA Replication 
DNA duplex (lagging strand), as a result of the 
geometry of the DNA helix, must be replicated in 
fragments. Synthesis of these fragments requires 
that the polymerase subunit be used over and 
over every 1-2 seconds by repeatedly delivering 
it from a finished fragment to a new one. Studies 
on the mechanism of rapid polymerase delivery 
-j;o new primed templates were supported by a 
grant from the National Institutes of Health. 
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 
form a twin polymerase. Since the chromosome 
has two strands of DNA, both of which must be 
replicated, the twin polymerase likely serves the 
function of coordinately replicating both DNA 
strands at the same time. These studies on the 
dimeric polymerase were also supported by a 
grant from the National Institutes of Health. 
These studies on four (7, 5, /?, t) of the eight 
accessory proteins of E. coli DNA polymerase III 
have been greatly aided by having the genes (the 
informational area in the DNA) for three of them 
(7, fi, r). This has provided large quantities of 
proteins for studies via molecular cloning and 
overproduction techniques. We have recently dis- 
covered the genes for each of the remaining five 
proteins (5, 5', x, 0) of DNA polymerase III and 
have used these genes to produce and purify large 
quantities of these proteins. Identification of the 
genes encoding 6', x, 4^, and 6 was supported by a 
grant from the National Institutes of Health. 
Toward a goal of understanding the overall 
structure of the holoenzyme, we are using the 
pure subunits to define the various subunit con- 
tacts within the holoenzyme and to assemble the 
entire replicating machine from its separate 
parts. Our plans to identify the individual func- 
tion of each subunit include biochemical analysis 
and also use of the newly discovered genes for the 
holoenzyme subunits to construct genetic mu- 
tants of E. coli for further clues to the function of 
each subunit. In addition to the polymerase holo- 
enzyme, the helicase and priming proteins are 
also central to the process of chromosome repli- 
cation, and we have initiated studies to examine 
how these proteins coordinate their actions with 
the holoenzyme. 
Model of the 13-clamp of the en- 
zyme DNA polymerase III. The 
function of the clamp is to 
tether the enzyme to the DNA, 
greatly accelerating DNA syn- 
thesis. The two parts of the ^- 
subunit dimer, represented in 
red and yellow, encircle the 
DNA ( in green and gray ), 
which has been modeled in the 
central cavity. 
From Kong, X.-P., Onrust, R., 
O'Donnell, M., and Kuriyan, J. 
1992. Cell 69:425-437. Copy- 
right © 1992 by Cell Press. 
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