Download 13 The Klenow fragment of DNA polymerase I Pol I actually appears

The Klenow fragment of DNA polymerase I
Pol I actually appears to be three enzymes in one polypeptide (polymerase, 3'→ 5'
exonuclease, 5'→ 3' exonuclease). Interestingly, mild digestion of Pol I with protease
(trypsin, subtilisn) cleaves the 109 kDa protein into two active fragments: A large Cterminal (Klenow) fragment (76 kDa) containing the polymerase and 3'→ 5' exonuclease,
and a small fragment that contains the 5'→ 3' exonuclease activity.
The separation of Pol I has practical consequences, because the Klenow fragment (KF) is
a very valuable enzyme in the laboratory. It can be used for cDNA synthesis, flushending restriction fragments with 5'-overhangs, and labeling DNA (random primer
method, etc.).
It is also important to note that, when mixed together, the large and small subunits of Pol
I can carry out nick translation. Besides being interesting from an evolutionary point of
view, this observation illustrates an important concept: exonuclease activities are not
always an intrinsic part of a polymerase. In some cases, exonucleases functionally
associate with the polymerase.
The structure of DNA polymerase I
As we have seen, the 5' → 3' exonuclease and polymerase activities act coordinately in
nick translation, even though they use different termini. It is easy to visualize how this
might occur- different termini are bound by different active sites on the enzyme, with the
5' → 3' exonuclease domain ahead of the Klenow fragment (KF) (Fig. 17). But what is
the relationship between 3'→ 5' exonuclease and polymerase, where both activities act on
the same terminus?
Insight into this and other important questions has come from analysis of the crystal
structures of KF and other polymerases. High-resolution analysis of the crystal structure
of KF (residues 324-928) shows that the polymerase domain resembles a right hand
gripping a double helix, with a separate juxtaposed exonuclease domain. (Ollis, D.L.,
Brick, P., Hamlin, R., Xuong, N.G., and Steitz, T. (1985) Structure of the large fragment
13
of DNA polymerase I complexed with dTMP. Nature 313: 762-766; Brautigam, C.A.
and Steitz, T.A. (1998) Current Opinion in Structural Biology 8: 54-63.)
The 3'→ 5' exonuclease domain
As noted above, KF can be divided into two domains (Fig. 17). The 3'→ 5' exonuclease
domain is the smaller of the two (amino acids 324-517). It is composed of 4 antiparallel
β
sheets surrounded by α helices. This domain contains a dNMP binding site, which
corresponds to the exonuclease active site. The 3'-OH can hydrogen bond with threonine
358. (ddNMPs can’t do this, which explains why ddNTPs bind poorly and are not
efficiently removed by the exonuclease.) Also, there is no apparent hydrogen bonding
between the dNMP purine and pyrimidine bases and the protein; this is expected because
all four dNMPs must fit into this site.
Mutants in the dNMP binding site lack
exonuclease activity but retain polymerase activity.
This confirms that the primer
terminus can be bound by two different sites in the Klenow fragment: the exonuclease
active site and the polymerase active site (formerly called the primer terminus site in Fig.
13).
The polymerase domain
The polymerase domain is the larger of the two, and binds both template primers and
dNTPs. When separately expressed, it has polymerase but not exonuclease activity. In
addition, mutations in amino acids that contribute to the polymerase catalytic site affect
polymerase activity but not exonuclease activity.
The polymerase domain encompasses amino acids 521-928. It is mainly helical, and its
most distinctive feature is a deep cleft ("palm") that is large enough to accommodate Bform duplex DNA (20-25 A wide and 25-35 A deep). The cleft is located between
helices I and O. A six-stranded β sheet forms the bottom of the cleft, and α helices form
the sides. One side is longer than the other and could curl over like “fingers”, the
opposite side resembles a “thumb”. Positive charge is distributed in the cleft, which
facilitates interaction with the negatively charged DNA backbone. As the DNA passes
14
through the crevice (palm), it enters from the direction of the exonuclease domain and is
bent by as much as 45° (see also Fig. 18).
Structural contributions to processivity
The thumb domain has been implicated in processivity (i.e. the ability of the enzyme to
remain associated with the template-primer over many catalytic cycles). At the tip of the
thumb, a disordered "flap" of about 50 amino acids that can close over the helix is
apparent. (The flap is between helices H and I). Closure of the flap apparently slows the
rate of DNA dissociation and contributes to processivity. In support of this idea, deletion
of the tip of the KF thumb reduces processivity by about a factor of four. Thus the flap is
sometimes called the “processivity domain.”
Structural contributions to catalysis (nucleotidyl transfer) and fidelity- a
stepwise mechanism of DNA synthesis
The active site residues lie in palm domain. The primer terminus is believed to interact
with tyrosine 776 (Y776). In addition, two essential aspartate residues, D705 and D882,
also lie at the base of the palm. As we shall see later, it is these acidic residues that bind
the Mg 2+ ions that are essential for catalysis. The second substrate, dNTP, is bound by a
pocket in the fingers domain. Interestingly, there are no hydrogen bonds formed between
the enzyme and the new base pair formed by the template and the incoming nucleotide.
This is useful, because there are four different types of nucleotides that must enter the
dNTP site.
But how does the enzyme discriminate between a correct incoming
nucleotide and an incorrect one?
The fidelity of a polymerase, its ability to discriminate between correct and incorrect
dNTPs, is obviously critical. As we have just learned, however, discrimination is not
based on interactions with individual bases. Instead, the correct addition (and error
discrimination) depends on relative nucleotide affinity and ultimately is based on shape.
Consider:
• Since there are no base-specific interactions between the enzyme and dNTP, all four
have an equal opportunity to enter the dNTP site
15
• However, those that can form a Watson-Crick base pair have a higher affinity for the
template base than those that do not. Hence they are more likely to be incorporated.
• A correct Watson-Crick base pair has the correct shape (steric complementarity), and
"fits" better than a mispair.
To see this more clearly, we can break the polymerase reaction down into a series of
steps, some of which are associated with changes in the shape of the enzyme. The
multistep process provides several opportunities to discriminate between correct and
incorrect nucleotides.
• The initial step involves binding the template-primer (to form a binary complex). This
results in a conformational change in which the thumb closes down around the DNA.
• Binding of the correct dNTP forms a productive polymerizing complex (a ternary
complex). This favors the correct nucleotide by a factor of 102 to 103.
• Once a dNTP is bound, another conformational change occurs in which the fingers
rotate toward the palm. This moves the correct dNTP closer to the active site and into
position for catalysis. This strongly favors a correct Watson-Crick base pair, as only
these have the correct fit. This has the effect of increasing polymerase fidelity to about
104 to 105. The closing of the fingers may be the rate limiting step.
• After polymerization the fingers open again to release pyrophosphate and permit entry
of another dNTP. The template-primer translocates and rotates relative to the enzyme so
that the new primer terminus is in the primer-binding site.
Adding an incorrect base greatly decreases the rate of polymerization, primarily because
the primer terminus is efficiently bound to the primer binding site only when it is
properly base paired. This give the 3'→ 5' exonuclease activity a chance to act on the
primer terminus. This editing function further increases fidelity by a factor of about 102.
What is the relationship between the polymerase and exonuclease active sites?
Structural data indicates that the 3'→ 5' exonuclease and polymerase activities reside on
separate domains of KF, and are about 30 A apart. Yet both the polymerase active site
and the exonuclease active site compete for the same primer terminus. So DNA binding
16
must differ in the polymerase and exonuclease reactions. (Breese. L.S., Derbyshire, V.,
Steitz, T.A. (1993) Structure of DNA polymerase I Klenow fragment bound to duplex
DNA. Science 260: 352-355)
A hypothesis to explain this suggests that mispairing stimulates exonuclease activity by
leading to partial melting of duplex DNA (~3 bp or so) (Fig. 18). DNA bending would
further help to destabilize the helix and help shift the equilibrium toward ssDNA in a
mispaired duplex. Melting seems to be important, because while the palm cleft can
accommodate a duplex, the crevice leading to the exonuclease site isn’t big enough and
can only accept ssDNA. Conversely, proper base pairing favors interaction between the
primer terminus and the polymerase active site.
The physiological role of Pol I
Is DNA polymerase I the major replicative polymerase of E. coli? For more than 10
years, this was thought to be the case; however, there were some problems:
•
Pol I is not processive enough (~ 200 NT on a gapped DNA template).
•
Pol I turnover number is too low (only ~ 600 nt/min/mol; or 10 nt/sec). The
replication fork in E. coli moves at roughly 1,000 nt/sec. at 37°. (Based on 4.7 x 106
bp, and a known chromosomal replication time of ~40 min.)
The most important challenge arrived with the isolation of E. coli mutants severely
deficient in Pol I (in 1969). Since then, several mutants have been isolated (the original
was called polA). All viable polA mutants make some residual amount of enzyme, and
all have 5’→ 3’ exonuclease activity. The original polA mutants grow at a normal rate,
but are very susceptible to UV irradiation and other agents that damage DNA. This
indicates a role for Pol I in DNA repair (more later).
But it also turns out that Pol I is essential for chromosomal replication -- it plays an
important role in primer removal and joining of nascent DNA fragments (Okazaki
fragment processing; more later). This is why all viable polA mutants have at least some
17
function, and more precisely why all have 5'→ 3' exonuclease activity. Both of these
processes (repair and primer removal) require 5'→ 3' exonuclease activity.
Therefore, Pol I is an essential enzyme, even if not the major replicative polymerase. Pol
I mutations that destroy polymerase activity are not lethal. (Presumably another pol can
substitute.) But Pol I mutations that destroy 5'→ 3' exonuclease activity are lethal.
Another Pol I family enzyme- T7 DNA polymerase
T7 is an E. coli bacteriophage (virus) that encodes its own DNA polymerase. Phage T7
polymerase is a small, single polypeptide enzyme (gp5, ~80 kDa) that contains a highly
active 3'→ 5' exonuclease activity in addition to a 5'→ 3' polymerase activity. It is the
enzyme responsible for replicating the ~35 kbp dsDNA phage genome.
A 5'→ 3' exonuclease activity is encoded by another phage gene. Its product (gp6) can
associate with T7 pol during replication. The gp 6 protein shows strong homology to the
5'→ 3' exonuclease domain of Pol I. So T7 is an example of a divided Pol I type enzyme;
it has separate polypeptides which provide the Klenow (5'→ 3' polymerase and 3'→ 5'
exonuclease) and 5'→ 3' exonuclease activities.
T7 pol is structurally homologous to the KF of Pol I and like Pol I, it is not a very
processive polymerase. However, a high degree of processivity is required to rapidly
duplicate the relatively large phage genome. How is this achieved?
The answer this question is quite surprising. In the cell, T7 pol exists in a tight 1:1
complex with thioredoxin (12 kDa). Thioredoxin is a cellular protein that normally
functions as a redox coenzyme for ribonucleotide reductase, an enzyme system that
synthesizes deoxyribonucleotides from ribonucleotide diphosphates.
In the T7 system, thioredoxin is recruited as a processivity factor. In a complex with T7
pol, thioredoxin clamps pol to the template primer, resulting in a 50-fold increase in
activity and about a 1,000-fold increase in processivity. It does this without binding
18
DNA, and without the help other accessory factors. How this unique association of
cellular and viral proteins came to be is not clear, but we now know how it works.
Recently, the structure of T7 pol complexed with a template primer, dNTP, and E. coli
thioredoxin was solved. As expected, the enzyme is quite similar to Pol I in structure,
although some important information regarding dNTP binding and polymerase
translocation were obtained from this study.
Most interestingly, it was found that
thioredoxin associates with a flexible loop extending from the thumb region (the
processivity domain) of the polymerase. This suggested that the thumb and thioredoxin
could swing across the DNA binding groove to encircle the template primer- essentially
forming a lid over the active site that functions like a clamp (Fig. 19). So the thioredoxin
clamp converts a Pol I-like enzyme into a highly processive, replicative polymerase.
(Doublie, S., Tabor, S., Long, A., Richardson, C.C., and Ellenberger, T. (1998) Crystal
structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution. Nature
391: 251-258.)
Further support for this mechanism has come from genetic and biochemical studies in
which the thumb region of T7 pol was grafted onto the tip of KF. These studies showed
that the T7 thumb could confer a thioredoxin-dependent increase in KF processivity
(Bedford, E., Tabor, R., and Richardson, C.C. (1997) The thioredoxin binding domain of
bacteriophage T7 DNA polymerase confers processivity on the Escherichia coli DNA
polymerase I. Proc. Natl. Acad. Sci. USA 94: 479-484.)
The T7 pol-thioredoxin (gp5-trx) complex is highly active and processive and today is
widely used for the Sanger sequencing reaction (in preference to Klenow). A modified
form of the enzyme, minus the 3'→ 5' exonuclease domain, is marketed commercially (as
"Sequenase"). T7 polymerase has several advantages over the Klenow fragment for
DNA sequencing:
•
Its high processivity allows it to read through secondary structure in the template
better than Klenow.
19
•
It better tolerates nucleoside analogues (dITP vs. dGTP) that can improve
electrophoretic resolution in sequencing gels.
Another Pol I family enzyme- Thermus aquaticus DNA polymerase
Another prokaryotic cellular (but non-E. coli) DNA polymerase of interest comes from T.
aquaticus. This polymerase is simply known as Taq polymerase, and is widely used for
PCR. Its utility for PCR stems from the fact that Taq polymerase is extremely heat stable
and can tolerate the high temperatures required to melt duplex DNA during PCR cycling.
This is because T. aquaticus lives in hot springs and thus must be able to replicate its
DNA at very high temperatures, in some cases above the TM of the duplex. Taq
polymerase (94 kDa protein) is truly extremely heat stable. It has a half-life of ~45 min
at 95°. The structural basis of this heat stability is still unclear.
Taq polymerase is a relatively processive enzyme, even though it is not known to
associate with a clamp. How this is achieved is also not clear, but it not a replicative
polymerase.
Taq pol is related to Pol I and like Pol I it has an intrinsic 5'→ 3'
exonuclease activity. However, it lacks a 3'→ 5' exonuclease activity and is therefore
error-prone.
Structurally, Taq polymerase shows considerable homology to Pol I and T7 polymerases
in the cleft (palm) region and its 5'→ 3' exonuclease domain also resembles that of Pol I.
However, the 3'→ 5' exonuclease domain, while present, is dramatically altered and nonfunctional. (Eom, S.H., Wang, J., and Steitz, T.A. (1996) Structure of Taq polymerase
with DNA at the polymerase active site. Nature 382: 278-281.)
The error-prone nature of Taq polymerase can sometimes cause problems in PCR. For
this reason, several other heat stable DNA polymerases which have proofreading
activities are commercially marketed.
Pyrococcus woesei
Two examples are Pwo polymerase from
and Pfu polymerase from P. furiosus (both are thermophilic
archaebacteria).
20
Other E. coli DNA polymerases
DNA Polymerase II
In wild type cells, Pol I activity is high and masks other polymerase activities. So, polA
mutants were searched for additional polymerases. Two new activities were found.
Later they were found in wild type cells as well. The first of the new polymerases
isolated from E. coli was called Polymerase II. Pol II differs from Pol I in that it lacks a
5'→ 3' exonuclease activity, and cannot use a nicked duplex template.
The structural gene for this Pol II is called polB. polB mutants grow normally and are not
sensitive to UV irradiation. However, there is evidence that Pol II is involved in DNA
damage induced SOS repair.
DNA Polymerase III
Pol III proved difficult to isolate and purify due to its complexity, instability, and low
number of copies per cell (10-20).
It is the most complex of the E. coli DNA
polymerases. Unlike Pol I and Pol II, it is not a single polypeptide but is composed of
several subunits. This is typical of replicative polymerases, and Pol III will serve as a
model for polymerases that duplicate cellular chromosomes. Pol III is the replicative
polymerase in E. coli and the catalytic subunit is the product of a gene known to be
essential for DNA replication: polC (a.k.a. dnaE).
The catalytic core of pol III (~167 kDa) is a complex of three proteins, which are very
tightly associated:
•
α,
the polymerase subunit (polC/dnaE gene product) -- 130 kDa
•
ε,
the 3'→ 5' exonuclease subunit (dnaQ/mutD gene product) -- 28 kDa
•
θ,
weakly stimulates ε -- 10 kDa
Mixing α and ε (in vitro) generates a 1:1 complex in which polymerase activity is
increased ~ 2-fold and 3' → 5' exonuclease activity is increased 50 to 100-fold. Also, only
when complexed with α does ε have significant activity on dsDNA templates. Clearly,
21
the ability of the core polymerase to proofread depends upon the association of ε with α.
The activities of ε and α appear to interact as they do in Pol I.
Pol III holoenzyme
However, Pol III (core) by itself clearly is not the replicative polymerase activity either.
The discovery of the replicative polymerase, Pol III holoenzyme, was driven by the
observation that neither Pol I, Pol II, or Pol III core are capable of replicating viral
ssDNA circles >1,000 nt long (i.e. all exhibit low processivity; Pol I ~10-20 nt, Pol III
core ~10 nt). Further, these activities have low turnover rates in vitro (Pol I ~10 nt/sec,
Pol II ~0.5 nt/sec, Pol III core ~20 nt/sec). Recall that the E. coli chromosome is a
circular DNA of some 4.7 x 106 bp, and can be replicated in 40 minutes. This predicts a
polymerase activity with a high turnover rate (~1,000 nt/sec) that is highly processive.
So, some other activity had to be responsible for replicative synthesis.
Eventually, an activity was found which was very efficient at replicating long templates.
This activity turned out to be Pol III core with several additional subunits that "clamp"
the core to the template and endow it with high processivity. Pol III holoenzyme is
highly processive on primed ssDNA coated with single-stranded DNA binding protein
(SSB)- processivity is in the megabase range (>105). (SSB helps to remove secondary
structure from the template.)
Pol III holoenzyme also has a very high turnover number (>750 nt/sec at 37° in vitro),
which is consistent with the rate of fork movement in E. coli. Because initiation is the
rate limiting step, this rapid rate of synthesis results from the high processivity of
holoenzyme.
The main function of Pol III holoenzyme is duplication of the E. coli chromosome,
although it has some function in repair. The holoenzyme shares many features, as well as
amino acid sequence homology, with the replicative polymerases of other prokaryotes
and their viruses (phages), as well as the replicative polymerases of eukaryotes and their
22
viruses. Thus it is a very good model for studying replicase activities. The shared
features of replicase enzymes include:
•
a multisubunit structure
•
requirement for ATP to clamp tightly to DNA (ATP is only needed to clamp
holoenzyme to the template; after that DNA synthesis is rapid and processive without
additional ATP)
•
very high processivity
•
rapid rate of DNA synthesis
(Kelman, Z., and O’Donnell, M. (1995) DNA polymerase III holoenzyme: Structure and
function of a chromosomal replicating machine. Ann. Rev. Biochem. 64: 171-200.)
23