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The chemistry of DN synthesisnd
The mode of action of DN polymerase
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For the synthesis of DNA to proceed, two key substratesfor the DNA polymerase must be present:
First, the four deoxynucleoside triphosphates- dCTP,
dGTP, dATP & dTTP.
dNTP
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Second, A particular arrangement of ssDNA and dsDNA called a
primer : template junction.
Primer : Template junc t ion
Formally, only the primer portion of the primer:template junction is a
substrate for DNA polymerase, since only the primer is chemically
modified during DNA synthesis.
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The addition of a nucleotide to a growing polynucleotide chain of
length nis indicated by the following reaction:
dNTP + (NMP)n (NMP)n+1 + P~P
pyrophosphate
The free energy of this reaction is small ( G = - 3.5 kcal/mole).
What then is the driving force for the polymerization of nucleotides
into DNA?
G is the change in free energy of a reacting system. It is the
portion of the total energy change in a system that is available
for doing a work (i.e. it is the useful energy).
DNA polymerase
The addition of pyrophosphate is the driving force for DNA synthesis
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If G is negative in sign, the reaction proceeds spontaneously with loss
of energy.
If G is positive in sign, the reaction proceeds only if free energy isgained (e.g. coupling with ATP).
If G is zero, the reaction system is at equilibrium and no net change
takes place.
Additional free energy is provided by the rapid hydrolysis of the
pyrophosphate into two phosphate groups by an enzyme known as
pyrophosphatase:
P~ P 2 Pi
pyrophosphatase
G = - 3.5 kcal/mole
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The net result of nucleotide addition and pyrophosphate hydrolysis is the
breaking of two high-energy phosphate bonds. Therefore, DNA synthesis
is a coupled process, with an overall reaction of:
NTP + (NMP)n (NMP)n+1 + 2 Pi
This is a highly favorable reaction with a G of -7 kcal/mole which
corresponds to an equilibrium constant (Keq) of about 105.
Such a high Keq means thatthe DNA synthesis is effectively irreversible.
[Keq = concentration of products/ concentration of reactants]
DNA polymerase
pyrophosphatase
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Mechanism of DNA Syn thesis
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The mechanism of DNA polymerase
Unlike most enzymes, which have an active site dedicated to a single
reaction, DNA polymerase uses a single active site to catalyze the
addition of any of the four dNTP.
DNA polymerase accomplishes this catalytic flexibility by the use of
the nearly identical geometry of the A:T and G:C base pairs (favorable
alignment of the substrate).
Each of the four bases exists in two alternative tautomericstates,
which are in equilibrium with each other.
The equilibrium lies far to the side of the conventional structures whichare the predominant states and the ones important for base pairing.
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The capacity to fo rm an alternat ive tautomer is a frequent
source of errors dur ing DNA synthesis.
The nitrogen atoms attached to the purine and pyrimidine rings are in the
aminoform in the predominant state and only rarely assume the imino
configuration.
Likewise, the oxygn atoms attached to the guanine and thymine normallyhave the ketoform and only rarely take on the enolconfiguration.
Tautomerizat ion of cy tosin e into the
imino form (a) and gu anine into the
enol form (b)
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Only when a correct base pair is formed are the 3OH of the primer
and the a-phosphate of the incoming dNTP in the optimum position for
catalysis to occur.
Incorrect base-pairing leads to dramatically lower rates of nucleotide
addition due to a catalytically unfavorable alignment of these
substrates.
This is an example of kinetic selectivity, in which an enzyme favors
catalysis using one of several possible substrates by increasing the
rate of bond formation only when the correct substrate is present.
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a-Attack of a primer 3OH end on a correctly base-paired dNTP.
b- The incorrect A:A base pair displaces the a pho sphate of theincom ing dNTP. This inco rrect alignm ent reduces the rate of
catalysis.
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DNA polymerase shows an impressive ability to distinguish between
ribo- and deoxyribonucleoside triphosphates. This discrimination is
mediated by the steric exclusion of rNTPs from the DNA polymeraseactive site.
In DNA polymerase, the nucleotide binding pocket is too small to
allow the presence of 2OH on the incoming nucleotide.
This space is occupied by two amino acids (discriminator amino
acids) that make van der Walls contact (a type of non-covalent
bonding) with the sugar ring.
Changing these amino acids to others with smaller side chains (e.g.
changing glutamate to an alanine) results in a DNA polymerase withsignificantly reduced discrimination between dNTPs and rNTPs.
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a.binding of a correctly base-paired dNTP to the DNA polymerase. Under
these conditions, the 3OH of the primer and the a-phosphate of the
dNTP are in close proximity.b.addition of a 2OH results in a steric clash with the discriminator amino
acids in the nucleotide binding pocket. This results in the a-phosphate of
the dNTP being displaced and a misalignment with the 3OH of the
primer, dramatically reducing the rate of catalysis.
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The three-dimensional structure of DNA polymerase
A molecular understanding of how the DNA polymerase catalyzes
DNA synthesis has emerged from studies of the atomic structure of
various DNA polymerases bound to primer:template junctions.
Based on the analogy to a hand, the three domains of the
polymerase are called the thumb, f ingersand palm.
Schemat ic o f DNA polymerase bound to a pr imer : temp late junct ion
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The palm domain is composed of a b sheet and contains the primary
elements of the catalytic site. The fingers and the thumb are composed
of a helices.
The palm domain of DNA polymerase binds two divalent metal ions,
typically Mg 2+or Mn2+that alter the chemical environment around thecorrectly base-paired dNTP and the 3OH of the primer.
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Two metal ions b oun d to DNA polymerasecatalyze nuc leot ide addit ion .
Metal ion A interacts with the 3OH resul t ing in reduced associat ion
between the O and the H. This leaves a nucleoph i l ic 3O- .
Metal ion B in teracts w i th the tr iphosp hate of the incom ing dNTP to
neut ralize their negative charge.
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Role of DNA polymerase palm domain
In addition to its role in catalysis, the palm domain also monitors the
accuracy of base-pairing of the most recently added nucleotides.
This region of the plymerase makes extensive hydrogen bond contacts
with base pairs in the minor groove of the newly synthesized DNA.
These contacts are not base-specific but only form if the recently added
nucleotides are correctly base-paired.
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Role of the DNA polymerase fingers domain
The fingers domain associates with the template region, leading to a
nearly 90turn of the phosphodiester backbone of the template
immediately after the active site.
This bend serves to expose only the first template base after the primer atthe catalytic site.
This conformation of the template avoids any confusion concerning which
template base is ready to pair with the next nucleotide to be added.
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DNA polymerase grips the template and the incoming
nucleot ide when a correct base pair is made
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Role of the DNA polymerase thumb domain
In contrast to the fingers and the palm, the thumb domain is not
involved in catalysis.
The thumb interacts with the recently synthesized DNA, this serves
two purposes:
a- It maintains the correct position of the primer and the active
site.
b- maintain a strong association between the DNA polymerase
and its substrate.
This association contributes to the ability of the DNA polymerase to
add many dNTPs each time it binds a primer:template junction.
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The path of the template DNA through
the DNA po lymerase
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