MOLECULAR BIOLOGY
Lecture 7
DNA POLYMERIZATION
• Understand the chemistry of DNA synthesis
• Understand the mechanism of catalysis by DNA polymerase
• Understand the proof reading function of DNA polymerase
CHEMISTRY OF DNA SYNTHESIS
• DNA synthesis requires deoxynucloside triphosphate (dNTPs)
• Primer: template junction
• Each of the dNTPs (dCTPs, dGTPs, dATPs and dTTPs) contain 3
phosphoryl groups which are attached to the 5’ hydroxyl of the 2 prime
deoxyribose
• The phosphoryl group close to ribose is Alpha phosphate middle group
beta phosphate and outer most group gamma phosphates
PRIMER: TEMPLATE JUNCTION
• As suggested by name, Primer: template junction has two key components
• The template provides ssDNA to be copied
• Primer provides 3’ free OH that will be extended as new nucleotides are
added.
• Primer portion of the primer template junction is the substrate for DNA
synthesis.
DNA SYNTHESIS
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DNA is synthesized by extending the 3’ end of the primer
3’ OH of primer attack α-phosphoryl group of dNTPs
Leaving group for this reaction pyrophosphate (Beta and Gamma phosphoryl
group)
The free energy change is rather small however additional free energy to drive
the reaction is obtained by the rapid hydrolysis of pyrophosphate by the help
of enzyme pyrophosphatase in to two phosphate groups.
The process can then be repeated with the 3’ OH of the newly added dNTP
serving as the nucleophile
The chemistry of DNA synthesis requires the formation of DNA in a polar
fashion
In biological polymerization the DNA is always formed by extending the 3’
end of the primer strand.
Template strand directs which dNTP is added through base pairing
THE MECHANISM OF DNA
POLYMERASE
• DNA polymerase?
• Monitors the ability of incoming nucleotide to form AT and GC base
pairing
• The synthesis of DNA is catalyzed by an enzyme called DNA polymerase
• The DNA substrate sits in large cleft of the DNA pol. that resembles to a
partially closed right hand
• Three domains of the polymerase i.e. Palm fingers and thumb domains
• The palm domain contains the active site for DNA synthesis
• What active sites actually does?
BASE PAIRING
• Correct base pairing is also required for catalysis
• Alpha phosphoryl group dNTP cannot properly align with the 3’ OH of
the primer strand
• Incorrect base pairing dramatically lower the rate of addition of
nucleotides due to catalytic unfavorable environment
• This is an example of kinetic selectivity?
• Process in which enzyme favors catalysis using one of the several possible
substrates by dramatically increasing the rate of bond formation only when
correct substrate is present.
• Once correct base pairing is done the reaction can continue
METAL IONS ASSOCIATED WITH
DNA POLYMERASE
• The palm domain also bind two divalent metal ions A and B (typically
Mg+2 Zn +2)
• These ions are crucial for DNA polymerization activity
• When the correct base pairing is done b/w the incoming dNTP and the
template. The finger domain move to enclose dNTP
• This conformational change bring the metal ions in the correct position to
function
• The metal A helps to deprotonate the 3’ OH of the primer producing and
oxyanion to attack the incoming Alpha phosphate of the incoming dNTPs
• Metal ion B interact the leaving group of pyrophosphate to neutralize the
charges
• Lysine and Arginine residues helps to stabilize the pyrophospahtes
• A tyrosine residues makes a staking interaction on the base with the base
of dNTP and holds dNTP in place for catalysis
• The finger domain is also plays a second role
• The finger domain associated with the template region leading to the 90
degree turn of the phosphodiaster backbone immediately after the active
site
• Prevent the confusion of addition of nucleotide addition
• Thumb domain is not directly involves in catalysis and it interacts which
has been most recently formed
• Hold on the primer template junction in the active sites
• Reduces the rate of dissociation
PROOF READING
• Palm domain hydrogen bonds to base pairs in minor grove
• Hydrogen bonds will only form if nucleotides are correctly base paired
• Mismatched base pairing the disturbed geometry of the substrate slow
downs the replication.
• The primer template junction is free to move at exonuclease site
• Removal of incorrectly base paired nucleotides from 3’ end hece called
DISCUSSION