1. BETA CLAMP AND PROGRESSIVE POLYMERASES
A DNA clamp, also known as a sliding clamp, is a protein fold that serves as a
processivity-promoting factor in DNA replication.
 As a critical component of the DNA polymerase III holoenzyme, the clamp protein binds
DNA polymerase and prevents this enzyme from dissociating from the template DNA
strand.
 The clamp-polymerase protein–protein interactions are stronger and more specific than
the direct interactions between the polymerase and the template DNA strand; because
the rate-limiting step in the DNA synthesis reaction is the association of the polymerase
with the DNA template, the presence of the sliding clamp dramatically increases the
number of nucleotides that the polymerase can add to the growing strand per
association event.
 The presence of the DNA clamp can increase the rate of DNA synthesis up to 1,000-fold
compared with a nonprocessive polymerase.
STRUCTURE
 The DNA clamp fold is an α+β protein that assembles into a multimeric structure that
completely encircles the DNA double helix as the polymerase adds nucleotides to the
growing strand.
 The DNA clamp assembles on the DNA at the replication fork and "slides" along the DNA
with the advancing polymerase, aided by a layer of water molecules in the central pore
of the clamp between the DNA and the protein surface. Because of the toroidal shape of
the assembled multimer, the clamp cannot dissociate from the template strand without
also dissociating into monomers.
 The first indication for the toroid shape of the sliding clamps came from the study of the
b subunit of the E. coli replicase.
 The DNA clamp fold is found in bacteria, archaea, eukaryotes and some viruses.
 In bacteria, the sliding clamp is a homodimer composed of two identical beta subunits of
DNA polymerase III and hence is referred to as the beta clamp.
 In archaea and eukaryotes, it is a trimer composed of three molecules. The T4
bacteriophage also uses a sliding clamp, called gp45.
BACTERIAL BETA CLAMP
 The beta clamp is a specific DNA clamp and a subunit of the DNA polymerase III
holoenzyme found in bacteria.
 The -subunit “clamp” ensures that the polymerase stays on the DNA
 Two beta subunits are assembled around the DNA by the gamma subunit and ATP
hydrolysis; this assembly is called the pre-initiation complex.
 After assembly around the DNA, the beta subunits' affinity for the gamma subunit is
replaced by an affinity for the alpha and epsilon subunits, which together create the
complete holoenzyme.
 DNA polymerase III is the primary enzyme complex involved in prokaryotic DNA
replication.
 The gamma complex of DNA polymerase III, composed of γ,δ,δ',χ,ψ subunits, catalyzes
ATP to chaperone two beta subunits to bind to DNA.
 Once bound to DNA, the beta subunits can freely slide along double stranded DNA.
 The beta subunits in turn bind the αε polymerase complex.
Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA Page 1

2.  The α subunit possesses DNA polymerase activity and the ε subunit is a 3’-5’
exonuclease.
 The beta chain of bacterial DNA polymerase III is composed of three topologically non-
equivalent domains (N-terminal, central, and C-terminal). Two beta chain molecules are
tightly associated to form a closed ring encircling duplex DNA.
 DNA Polymerase III (pol III) from E. coli is a single protein of molecular weight 130 kDa
(130,000 grams per mole).
 It is also referred to as polC, dnaE, or the alpha subunit. Though the molecule has DNA
polymerase activity by itself, polIII works to replicate DNA in the bacterial cell in
conjunction with other proteins.
 This multi-protein complex is referred to as the pol III holoenzyme.
 The proteins (called subunits) that associate with pol III in the holoenzyme perform
several functions.
 The most interesting subunit is called beta, which forms a donut shaped ring around the
DNA and helps to anchor the holoenzyme to the DNA during replication.
 By acting as a sliding "clamp", beta helps the holoenzyme to replicate long stretches of
DNA without "falling off" the strand (this is called processivity).
 Pol III holoenzyme directs both leading and lagging strand synthesis simultaneously by
virtue of having two polymerase subunits.
 The Table summarizes the pol III subunits, subassemblies, and their functions:
DNA polymerase III subunits and subassemblies
Subunit Function Subassembly (complex)
alpha DNA polymerase
core (there are two cores per DNA polymerase
epsilon 3'-to-5' exonuclease (editing exonuclease)
III holoenzyme)
theta stimulates 3'-to-5' exonuclease
tau dimerizes cores, activates DnaB helicase activity
gamma binds ATP
delta unknown
delta prime stimulates clamp loading gamma complex (clamp loader), uses ATP
energy when loading beta onto primed DNA.
interacts with SSB to allow removal of DnaG primase
chi
from primer
psi unknown
The Sliding clamp. The beta subunit can be loaded onto
DNA by the clamp loader (gamma complex) in an ATP-
dependent reaction). (The clamp loader also unloads
clamps!) Beta cannot be loaded onto linear DNA ,
covalently closed circular DNA, or single-stranded
circular DNA, but it can be loaded onto nicked circles,
gapped circles, and primed single-stranded circles; that
is, clamp loader requires and recognizes a 3'-hydroxyl-
beta
terminus (primer-terminus). Once loaded onto a nicked
circle, beta stays associated with the DNA. However,
linearization of the nicked circle with a restriction
endonuclease releases beta from the DNA; that is, beta is
a sliding clamp. It can slide along double-stranded DNA
(or DNA-RNA in double-stranded form), but cannot slide
on single-stranded DNA or single-stranded DNA coated
with SSB.
Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA Page 2

3. Quick Comparison of DNA polymerases I and III
DNA polymerase III DNA polymerase I
DNA Pol III holoenzyme is an asymmetric dimer; DNA Pol I is a monomeric protein
i. e., two cores with other accessory subunits. It with three active sites. It is
can thus move with the fork and make both distributive, so having 5'-to-3'
Structure
leading and lagging strands. exonuclease and polymerase on the
same molecule for removing RNA
primers is effective and efficient.
Polymerization and 3'-to-5' exonuclease, but on Polymerization, 3'-to-5'
different subunits. This is the replicative exonuclease, and 5'-to-3'
polymerase in the cell. Can only isolate exonuclease (mutants lacking this
Activities conditional-lethal dnaE mutants. Synthesizes essential activity are not viable).
both leading and lagging strands. Primary function is to remove RNA
No 5' to 3' exonuclease activity. primers on the lagging strand, and
fill-in the resulting gaps.
250-1,000 nucleotides/second. This is as fast as 20 nucleotides/second. This is NOT
the rate of replication measured in Cairns' fast enough to be the main
experiments. Only this polymerase is fast replicative enzyme, but is capable of
Vmax (nuc./sec)
enough to be the main replicative enzyme. "filling in" DNA to replace the short
(about 10 nucleotides) RNA primers
on Okazaki fragments.
Highly processive. The beta subunit is a sliding Distributive. Pol I does NOT remain
clamp. The holoenzyme remains associated with associated with the lagging strand,
Processivity
the fork until replication terminates. but disassociates after each RNA
primer is removed.
10-20 molecules/cell. In rapidly growing cells, About 400 molecules/cell. It is
there are 6 forks. If one processive holoenzyme distributive, so the higher
Molecules/cell (two cores) is at each fork, then only 12 core concentration means that it can
polymerases are needed for replication. reassociate with the lagging strand
easily.
DNA polymerase III holoenzyme
(Note: no beta subunits are shown; without beta, this form of the complex is called DNA pol III)
Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA Page 3

4. Steps involved in loading a sliding clamp for processive DNA synthesis.

 The beta subunit of DNA polymerase-III holoenzyme confers upon the polymerase the
ability to faithfully track the rapidly moving replication fork while synthesizing leading
and lagging strand DNA simultaneously.
 The beta subunit, known as a sliding clamp, forms a stable ring-shaped structure that
encircles DNA.
 Once attached to the beta subunit, the catalytic alpha subunit of the polymerase can
move along DNA for tens of kilobases or more without dissociation, incorporating new
nucleotides into the growing DNA strand at speeds as high as 750 nucleotides per
second.
 The sliding clamps and their associated clamp loading systems are of broad importance
in many cellular processes involving DNA, beyond that originally imagined by their
discovery as essential factors for chromosomal replication.
Dr. Shiva C. Aithal, Dept. of Microbiology, Dnyanopasak College, PARBHANI (Maharashtra) INDIA Page 4