Genetics 
Four genes that encode five subunits of the ho- 
loenzyme have been known for some time. The 
dnaE gene encodes a, the DNA polymerase; dnaQ 
encodes e, the 3 -5' exonuclease; dnaN tncodes the 
/? subunit; and dnaX encodes both the r and 7 sub- 
units (7 is produced from dnaX by a translational 
frameshifting mechanism). Each of these genes is 
essential for cell viability, as expected for subunits 
of the chromosomal replicase. 
Dr. O'Donnell's laboratory has recently identified 
and sequenced the genes for the remaining five sub- 
units: b (holA), 8' (holB), x (holC), \p (holD), and 6 
(holE) . The studies on holB, holC, holD, and holE 
were supported by a grant from the National Insti- 
tutes of Health. These genes are now being used to 
determine the intracellular role of each subunit by 
constructing strains of E. co// that are missing one or 
more of these genes. Preliminary studies indicate 
that holE is not essential for cell viability. Work on 
the other genes is in progress. Strains that survive in 
the absence of a hoi gene will be analyzed for 
growth defects. It seems possible that a subunit (s) 
of the holoenzyme could perform a specialized role 
such as proofreading one particular mismatch or in- 
terfacing with other cellular machineries for DNA 
repair, recombination, or mutagenesis. Therefore 
any nonessential subunit genes will be studied in a 
variety of genetic backgrounds to identify pos- 
sible roles in fidelity, repair, recombination, or 
mutability. 
Biochemistry 
The holoenzyme is a remarkably rapid (750 nu- 
cleotides/s) and processive (> 100,000 nucleo- 
tides/template-binding event) polymerase. This 
rapid and processive synthesis sets it apart from re- 
pair polymerases and distinguishes it as a chromo- 
somal replicase. During the past two years the mo- 
lecular mechanism of this remarkable speed and 
processivity of the holoenzyme has been elucidated 
and is described below. 
The holoenzyme can be resolved into three com- 
ponents. One is a three-subunit subassembly called 
the core polymerase that contains the a (polymer- 
ase), € (3'-5' exonuclease), and B subunits. Another 
is the 7 complex which has five subunits (y5d'x4')- 
The third component is the ^ subunit. The core poly- 
merase is not a rapid and processive polymerase but 
instead is even slower and less processive than DNA 
polymerase I. The 7 complex and /3 are both needed 
to confer the processive speed onto the core poly- 
merase. They do so in the following fashion. The 7 
complex recognizes a primed template and then 
couples the energy of ATP hydrolysis to clamp a 
dimer of the /? subunit onto the primed template. 
After this the 7 complex may leave and the /3 dimer 
remains behind, bound to the primed template. This 
P dimer remains tightly bound to circular DNA, but 
upon linearizing the DNA, it slides freely off over 
either end. Hence the (3 dimer has mobility on DNA, 
and since it falls off linear DNA but not circular DNA 
it must bind the DNA by encircling it like a dough- 
nut. The 7 complex must function to assemble the ^ 
dimer around DNA. Recently, in collaboration with 
Dr. John Kuriyan (HHMI, Rockefeller University), 
the x-ray structure of the (3 dimer was solved. As 
expected, |8 is a ring-shaped protein with a cen- 
tral cavity of sufficient size to accommodate du- 
plex DNA. 
The |8 dimer, besides acting as a sliding clamp by 
encircling DNA, also binds directly to the core poly- 
merase. Hence the fundamental basis for high pro- 
cessivity is a sliding clamp of 13, which continuously 
holds the polymerase down to the primer template, 
thereby making it behave in a highly processive fash- 
ion. As the polymerase synthesizes DNA, it simply 
moves forward and pulls the /3 sliding clamp along 
with it. This project was supported by the National 
Institutes of Health, which is also supporting 
current investigations on how the 7 complex cou- 
ples ATP to the opening and closing of the (3 ring. 
"Form follows function," and both structural and 
functional studies are important complementary av- 
enues of investigation. Knowledge about the struc- 
ture of the holoenzyme, the stoichiometry of its sub- 
units, and the subunit-subunit contacts within it are 
important to understand how they perform their 
functions as a unit. To this end, all 10 subunits have 
been overproduced and purified. The a, t , (3, 7, and 
T proteins have been overproduced by molecular 
cloning of their genes in the laboratories of Drs. 
Arthur Kornberg and Charles McHenry and Drs. 
Harrison Echols, Alvin Clark, and James Walker. Like- 
wise, using the recently identified genes, Dr. 
O'Donnell's laboratory has overproduced and puri- 
fied the 5, 6', X. 'A- and d subunits. Using these pure 
proteins, they have identified many subunit contacts 
and determined many stoichiometrics, although the 
study is not yet complete. Among these contacts is 
one mediated by the r subunit, which is a dimer in 
its native state. The r dimer binds directly to the a 
subunit, the DNA polymerase. Since t is dimeric, it 
binds two polymerase subunits. Hence, as predicted 
long ago by Dr. Kornberg, the holoenzyme has two 
DNA polymerase subunits, as expected for a repli- 
case that must synthesize both strands of a duplex 
DNA chromosome. 
CELL BIOLOGY AND REGULATION 99 
