The chemistry of DNA synthesis And

The mode of action of DNA polymerase

 For the synthesis of DNA to proceed, two key substrates for the DNA polymerase must be present:

First, the four deoxynucleoside triphosphates- dCTP, dGTP, dATP & dTTP.

dNTP

 Second, A particular arrangement of ssDNA and dsDNA called a primer : template junction.
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.

Primer : Template junction

The addition of pyrophosphate is the driving force for DNA synthesis

The addition of a nucleotide to a growing polynucleotide chain of length n is indicated by the following reaction:

DNA polymerase

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).

 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 is gained (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:
pyrophosphatase

P~ P
G = - 3.5 kcal/mole

2 Pi

 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:
DNA polymerase

NTP + (NMP)n
pyrophosphatase

(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 that the DNA synthesis is effectively irreversible. [Keq = concentration of products/ concentration of reactants]

Mechanism of DNA Synthesis

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 tautomeric states, which are in equilibrium with each other. The equilibrium lies far to the side of the conventional structures which are the predominant states and the ones important for base pairing.

 The nitrogen atoms attached to the purine and pyrimidine rings are in the

amino form in the predominant state and only rarely assume the imino configuration.
Likewise, the oxygn atoms attached to the guanine and thymine normally have the keto form and only rarely take on the enol configuration.

Tautomerization of cytosine into the imino form (a) and guanine into the enol form (b)

The capacity to form an alternative tautomer is a frequent source of errors during DNA synthesis.

 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.

a- Attack of a primer 3OH end on a correctly base-paired dNTP. b- The incorrect A:A base pair displaces the a-phosphate of the incoming dNTP. This incorrect alignment reduces the rate of catalysis.

 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 polymerase active 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 with significantly reduced discrimination between dNTPs and rNTPs.

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.

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, fingers and palm.

Schematic of DNA polymerase bound to a primer:template junction

 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 the correctly base-paired dNTP and the 3OH of the primer.

Two metal ions bound to DNA polymerase catalyze nucleotide addition. Metal ion A interacts with the 3OH resulting in reduced association between the O and the H. This leaves a nucleophilic 3O- . Metal ion B interacts with the triphosphate of the incoming dNTP to neutralize their negative charge.

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.

Role of the DNA polymerase fingers domain

The fingers domain associates with the template region, leading to a nearly 90 turn 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 at the 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.

 The fingers are also important to catalysis, several residues (e.g. Tyr., Lys., & Arg.) located within the fingers bind to the incoming dNTP. Once a base-pairs correctly to the template DNA, the O-helix of the finger domain (open conformation) rotates by 40 (closed conformation). When the polymerase is in the closed conformation, the O-helix makes several important interactions with the incoming dNTP. A tyrosine makes stacking interactions with the base of the dNTP and the two charged residues associate with the triphosphate. The combination of these interactions positions the dNTP for catalysis mediated by the two metal ions (Mg 2+ or Mn2+ ).

DNA polymerase grips the template and the incoming nucleotide when a correct base pair is made

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.

The path of the template DNA through the DNA polymerase