DNA Technology Lect 2

8/12/2019 DNA Technology Lect 2

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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

Documents

DNA Technology Lect 2