DNA Polymerase I:
Klenow Fragment
By: Alonzo Knowles and Brendon Scicluna
Introduction to Pol I:
DNA Polymerase I is only one of three
possible DNA polymerases isolated from Escherichia coli. These three enzymes all
differ in molecular weight, ability to utilize different template-primer systems, salt
sensitivity, inhibition by sulfhydryl blocking reagents, and locus of structural genes.
Of the three DNA Polymerase I is the predominant polymerizing enzyme found in
E. coli. Its structure contains a single disulfide bond and one sulfhydryl group.
This relatively simple enzyme provides an excellent way of observing the
essential characteristics of a DNA synthesizing enzyme. DNA Polymerase I, or Pol I,
consists of a single polypeptide chain that is 102 kDa in size. It has a molecular
weight of roughly 109,000 g/mol and has an optimum pH of about 7.4. The enzyme is
unique in that it catalyzes three very distinct reactions. It has both DNA
polymerase activity as well as two different exonuclease activities (3' --> 5' as well
as 5' --> 3' exonuclease activities) associated with it.
Observe the cartoon structure of the Klenow fragment of DNA pol I by pressing the
button below.
Cartoon
Pol I is a major component of the DNA replication machine because of its
polymerase activity. It synthesizes new DNA, binding at a replication fork in DNA
(Y-shaped junctions in DNA) and uses the old strands of DNA as a template for synthesizing
new strands. The catalysis activity of the enzyme usually allows the addition of
nucleotides to the 3'-hydroxyl end of a new strand. Nucleosides
entering this reaction are initially energy-rich nucleoside triphosphates, which provide
energy for the polymerization process. It is the hydrolysis of the phosphoanhydride
link in the nucleoside triphosphate that provides energy for condensation reactions
that bonds nucleotide monomers to the chain and generates pyrophosphate. In this way
DNA polymerase couples the release of energy to a polymerization reaction. The
pyrophosphate undergoes further reduction to inorganic phosphate, which makes the reaction
effectively irreversible.
As DNA polymerase I synthesizes
the new DNA strand it does not dissociate from the strand as it adds nucleotides to it.
Instead it remains attached and moves along the DNA stepwise as it polymerizes.
The new DNA chains that are created are base paired with the template strands, also
the template strands and the new strand are antiparallel to one another. Since DNA
polymerases can only catalyze DNA growth in one direction (5' --> 3' direction) it is
necessary for it to synthesize the DNA discontinuously for one of the, this strand is
usually stitched together later and it is more commonly referred to as the lagging strand.
The other DNA strand, which is synthesized continuously, is termed the leading
strand.
The 3' à 5' exonuclease activity allows Pol I to perform
"proofreading" on newly synthesized DNA. The 3' à 5' exonuclease prevents
Pol I from adding wrong nucleotides to the growing DNA strands by removing mispaired
nucleotides. This proofreading step is essential to increasing Pol I's accuracy.
On the other hand, the 5' à 3' exonuclease activity allows Pol I to degrade the
DNA strand ahead o the advancing polymerase. This capability allows the Pol I enzyme
to remove and replace nucleotides in a strand simultaneously. This function is
extremely useful as Pol I seems to be mostly associated with DNA repair, where it is
essential that damaged DNA be destroyed and replaced.
Mouse Commands
Use the left mouse button to manipulate the screen
- Click the right button for a menu similar to the Quanta CHARMm draw
menu
- Hold down shift and use the left mouse button to zoom (up = out, down
= in)
- Hold down the control button and use the right mouse button to
translate the molecule on the axis.
The Klenow Fragment:
Observe the structure of the protein and note where the DNA binds to it by pushing the
button below.
Protein
One very important feature of Pol I is
its ability to be cleaved into two separate components by mild proteolytic treatment.
The smaller fragment is the one responsible for the 5' --> 3' exonuclease
activity of the enzyme. The larger of the two fragments (the Klenow Fragment) is
responsible for the polymerase and 3' --> 5' exonuclease (proofreading) portions of the
enzyme. Observe the two parts of the enzyme as indicated in the images shown
directly below:
The
Klenow fragment is indicated in red above and you can clearly see how it interacts with
the smaller fragment in DNA synthesis and repair. It has a molecular weight of
roughly 68,000 and it consists of two domains that are joined by only a single residue.
A view of the main backbone of the Klenow fragment can be observed by clicking the
button below:
Now observe the amino and carboxyl terminus ends of the protein; as the color shifts to
blue you are approaching the carboxyl terminus, as it shifts to red you are approaching
the amino terminus.
Termini
Upon
viewing of the secondary structure it is observed that the fragment consist of mostly
alpha helicies. More specifically, the entire protein consists of only four beta
sheets but contains approximately twenty-six alpha helicies. In the pictures below
images of both the alpha helixes and beta
sheets are given. The fragment contains only four beta sheets, which are
labeled in the image below independent of the helicies as A, B, C, and D. Beta sheet
(A) consists of only six strands in varying orientations; (B) and (C) both consist of two
strands, which are anti-parallel. The final sheet, sheet (D), contains four sheets
and is likewise anitparallel in nature.
The Klenow fragment also consists of: 0 beta barrels, 1 beta-alpha-beta
unit, 5 beta bulges, 5 beta hairpins, 8 gamma turns, and 32 beta turns.
Take a look at the overall secondary structure of this protein by pressing the button
below.
Structure
As
stated earlier the fragment can be separated into two domains. The structure of the
two domains of the fragment and the way that they interact is why shape of the Klenow
fragment, similar to most known polymerase structures, is likened to a hand. As
such, the large and small domains can also be referred to as the finger and thumb domains.
The image depicted below identifies the small and large domains of the Klenow
fragment and also indicates the joining point for the two fragments:
It is the larger of the two
domains that contains the polymerase enzymatic activity while the smaller domain is
responsible for the 3' --> 5' exonuclease activity. It is because of its
multi-functional nature that the Klenow fragment has two active sites contained within
these domains and the two active sites are in actuality separated by no more than 35
angstrom. The image below indicates the position of both active sites on the
fragment; they are labeled E and P, for exonuclease and protease activity respectively:
It is the proximity of the two active sites
that allows the enzyme to conduct both polymerization and exonuclease activity on DNA at
the same time. The Klenow fragment because of its unique purpose must be able to
bind the DNA template strand, the primer strand, and the dNTP all at the same time.
This makes the whole synthesis and repair process continuos and more
efficient. The actual process of binding involves only ten residues that are
actually situated very far apart in the primary structure of the molecule. These
residues are listed below:
Asn 675, Asn 678, Lys 635, Arg 631, Glu 611, Thr 609.
Arg 835, Asp 827, Ser 582, Asn 579.
It is the residues that are marked in red which
are actually involved in binding of the fragment to the primer strand during DNA synthesis
and repair. The residues, which are colored in green, are the ones that attach to
the template strand during the synthesis and repair process. It wasn't until 1988
that the importance of this region was identified when S. Basu et al., when they modified
lysine 635 with the aid of Pyridoxal 5' Phosphate and the observed result was a decrease
in DNA synthesis. In the following two images; the first image shows the Klenow
fragment, along with the residues when attached to DNA, the second image shows only the
individual residues listed above, isolated from the rest of the fragment:
1 
2
Similarly observe DNA bound in the binding cleft of the Klenow Fragment by pressing the
DNA/RNA button below.
DNA/RNA
For DNA binding and protein
activity to occur an three factors must be taken into consideration: (1) All four
deoxyribonucleosides must be available, (2) there must be a DNA template, and (3) a primer
strand must be attached for new DNA to be extended.
Push the Activator On/Off button to observe the Mg2+ and its binding with the Klenow
fragment.
Upon binding of DNA a conformational change
occurs in the protein. The thumb region is situated to the right of the cleft in the
image below. This region is made up of two alpha helicies connected by two smaller
helicies, two strands and a loop. When DNA binds to the protein the two helicies
move closer to the large cleft of the fragment. This change in protein conformation
is essential to allowing both the polymerase and exonuclease activities of the Klenow
fragment to interact with the same DNA strand. The images below clearly depict the
conformation changes that the protein undergoes during DNA binding:
The Klenow fragment is frequently used in
Biology, specifically Molecular Biology, to synthesize DNA when it is undesirable to
destroy the paternal strand. Good examples for uses of the Klenow fragment are
primer extension to determine the start of transcription, or to perform DNA sequencing.
In contrast the entire Pol I enzyme may be used to perform nick translation on 5'
--> 3' degradation of DNA.
References:
Alberts et al., "Essential Cell Biology"(Garland Publishing
Inc., NY, 1998), p.192-194
R.F. Weaver, "Molecular Biology"(Mcgraw-Hill Comp., Inc.,
1999), p. 651-653
http://www.worthington-biochem.com/manual/D/dnap.html
http://bssv01.lancs.ac.uk/StuWork/Bios31698/Klenowfragment/Fxgf.html
http://www.bimcore.emory.edu/Kins/CHEM441/Wthiel/dna/contents.html