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Chap 8 - DNA POLYMERISATION

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37 views3 pages

Chap 8 - DNA POLYMERISATION

Uploaded by

bharat
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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DNA POLYMERISATION

 In order for genetic information to be passed on from a parent cell to a daughter cell, the
parental DNA must be copied into two identical daughter DNA molecules.
 DNA synthesis requires deoxynucleoside triphosphates (dNTPs) and a primer- template
junction.
 Each of the four dNTPs ( dGTP, dCTP, dATP and dTTP) has three phosphoryl groups which are
attached via the 5’ hydroxyl of the 2’- deoxyribose. The phosphoryl group closest to the
ribose is the α- phosphate, the middle group is the β- phosphate, and the outermost group is
the γ- phosphate.
 The primer- template junction has two key components. The template provides the single-
stranded DNA to be copied. The primer provides a free 3’ hydroxyl at which a dNTP can be
added.
 The 3’ hydroxyl of the primer strand attacks the α- phosphoryl group of the incoming dNTP
in a nucleophilic substitution reaction. The leaving group for the reaction is pyrophosphate,
which is formed from the β- and γ- phosphoryl groups of the dNTP.
 The free energy change for this reaction is rather small. However, additional free energy is
provided by the rapid hydrolysis of the pyrophosphate into two phosphate groups. This
reaction is catalysed by the enzyme pyrophosphatase.
 The process can then be repeated, with the 3’ hydroxyl of the newly added dNTP serving as
the nucleophile. The chemistry of DNA synthesis requires that DNA is made in a polar
fashion. In biological polymerization, DNA is always made by extending the 3’ end of the
primer strand.
 The template strand directs which of the four possible dNTPs is added. During replication,
the dNTP that base pairs with the template strand is highly favoured for addition to the
primer strand.
 The main enzyme of DNA synthesis is DNA polymerase. The DNA substrate sits in a large cleft
of the DNA polymerase that resembles a partially closed right hand. Based on the analogy to
a hand, three domains of the polymerase called the palm, fingers, and thumb have been
described.
 The palm domain houses the active site for DNA synthesis. The active site of DNA
polymerase is able to distinguish between rNTPs and dNTPs, even though rNTPs are present
at approximately 10- fold higher concentration in the cell.
 The nucleotide binding pocket is too small to accommodate the presence of a 2’- hydroxyl
on the incoming nucleotide, allowing the polymerase to sterically exclude rNTPs.
 Correct base pairing is also required for catalysis. If an incorrect base pair forms, the α-
phosphoryl group of the dNTP cannot properly align with the 3’ hydroxyl of the primer
strand. Once the proper dNTP is bound in the pocket, the reaction can continue.
 The palm domain also binds two divalent metal ions. These metal ions are crucial for DNA
polymerization activity.
 Once a correct base pair is formed between the incoming dNTP and the template, the finger
domain moves to enclose the dNTP. This conformational change brings the divalent metal
ions into the correct positions to function.
 Metal ion A helps to deprotonate the 3’ hydroxyl of the primer, producing an oxyanion to
attack the α- phosphate of the incoming dNTP.
 Metal ion B coordinates the negative charges of the β- and γ- phosphates of the dNTP, and
stabilizes the pyrophosphate leaving group. Lysine and Arginine residues on the finger
domain also help to stabilize the pyrophosphate, and, through stacking interactions, a
tyrosine residue attached to the fingers helps to hold the dNTP in place for catalysis.
 The finger domain also plays a second role. The finger domain associates with the template
region, leading to a nearly 90o turn of the phosphodiester backbone of the template
immediately after the active site. This conformation of the template leaves only a single
template base in the active site, preventing any confusion as to which base is ready to pair
with the next nucleotide to be added.
 In contrast to the fingers and the palm, the thumb domain is not intimately involved in
catalysis. Instead, the thumb interacts with the DNA that has been most recently
synthesized. This serves to maintain the correct position of the primer- template junction in
the active site, and reduce the rate of dissociation of the polymerase from the DNA
substrate.
 Another function of the palm domain is proofreading. The palm region makes extensive
hydrogen bond contacts with the base pairs in the minor groove of the newly synthesized
DNA. These contacts are not base- specific, but they only form if the recently added
nucleotides are correctly base paired.
 In the rare event that a mismatched base pair is added, the distorted geometry of the
substrate causes the replication rate to slow dramatically. The palm domain is now not able
to make contacts with the minor groove, and consequently the primer- template junction is
now free to move about and interact with the exonuclease site.
 The exonuclease site removes incorrectly base- paired nucleotides starting from a 3’ DNA
end, and is therefore called a proofreading exonuclease. Removal of the incorrectly paired
base is a matter of kinetics with a mismatched base the diminished replication rate is now
slower than the exonuclease rate, and therefore excision of the mismatched base is greatly
favoured over polymerization on top of the mismatch. After this potential mutation is
corrected, the primer- template junction slides back into the DNA polymerase site, and the
replication continues.

Conclusion

 DNA synthesis requires deoxynucleoside triphosphates (dNTPs) and a primer- template


junction. The 3’ hydroxyl of the primer strand attacks the α- phosphoryl group of the
incoming dNTP. Additional free energy is provided by the rapid hydrolysis of the
pyrophosphate leaving group.
 The main enzyme of DNA synthesis is DNA polymerase. Due to its shape, three domains of
the polymerase called the palm, fingers and thumb have been described.
 The palm domain houses the active site for DNA synthesis. The palm domain can distinguish
between rNTPs and dNTPs. Two divalent metal ions in the palm domain are crucial for DNA
polymerization activity.
 When a correct base pair is formed between the incoming dNTP and the template, the finger
domain moves to enclose the dNTP. This conformational change brings the divalent metal
ions into the correct position to function. Lysine and Arginine residues on the finger domain
help to stabilize the pyrophosphate, and a tyrosine residue helps to hold the dNTP in place
for catalysis.
 The finger domain also associates with the template region, leading to a nearly 90 o turn of
the phosphodiester backbone of the template strand. The thumb domain interacts with
recently synthesized DNA, serving to maintain the correct position of the primer- template
junction, and reduce the rate of dissociation of the polymerase from the substrate.
 Another function of the palm domain is proofreading. When a mismatched base is added,
the replication rate slows dramatically, and the primer- template junction is free to interact
with the exonuclease site. The exonuclease site removes the mismatched base, and
replication continues.

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