Chemistry of DNA synthesis

To access and download PowerPoint presentation on 'DNA replication' click on the link below:
Download slides

Chemistry of DNA synthesis

Substrates for DNA synthesis:

•      Deoxyribonucleotide triphosphates (dNTPs)

•      Primer: template junction

The primer provides a free 3’-OH while template provides a single-stranded DNA to be copied.

DNA polymerization is a NUCLEOPHILIC SUBSTITUTION (S_N2) REACTION.

DNA polymerase has 3 main domains:

–     Palm domain

–     Thumb domain

–     Finger domain

v  Palm domain:

•      Possess the active site of DNA synthesis and is composed of a β-sheet.

•      The active site in the palm domain can distinguish between rNTPs and dNTPs.

•      The rNTPs are present in around 10 fold higher concentrations in the cell.

•      But the nucleotide-binding pocket is too small for the 2’-OH on the incoming rNTP.

•      Thus the polymerase can exclude the rNTPs by steric constraining.

•      Correct base pairing is also required for catalysis.

•      If an incorrect dNTP comes, its α-phosphoryl group cannot properly align with the 3’-OH of the growing strand.

•      Once the correct dNTP is bound in the pocket, the reaction can continue.

•      The palm domain also binds Zn²⁺ and Mg²⁺ which are crucial for catalysis.

v  Finger domain:

•      Composed of α-helix.

•      Once the correct dNTP is bound in the pocket, the finger domain moves to enclose the base-paired dNTPs.

•      This conformational change brings the dNTP and the primer (or growing DNA strand) into correct orientation with the divalent metal ions.

•      The O-helix of the finger domain moves 40° to enclose the base by stacking interaction with its tyrosine residue.

•      Metal ion A helps to deprotonate the 3’-OH of the primer producing an oxyanion.

•      This oxyanion attacks the α–phosphoryl group of the incoming dNTP.

•   Metal ion B coordinates the negative charge of the β- and γ-phosphate groups and stabilizes the pyrophosphate leaving group.

•      Lysine and arginine residues on the finger domain also help to stabilize the pyrophosphate and the tyrosine residue holds the dNTP in place for catalysis (stacking interaction).

•   The finger domain also associates with the template region resulting in a 90° turn in the template which helps to avoid confusion in the active site.

•      This ensures that only one template nucleotide remains in the active site.

v  Thumb domain:

•      It is not intimately involved in catalysis.

•   It interacts with the DNA that has been synthesized most recently and holds the primer: template junction in the active site.

•      This reduces the dissociation of the polymerase from the template.

Proofreading

•     If an incorrect nucleotide is incorporated by DNA polymerase (frequency of error is 10^-6), it is recognized immediately.

•      The 3’ → 5’ exonuclease activity of the polymerase then excise the incorrect nucleotide from the new strand.

•      The polymerase then resumes its forward motion and inserts the correct nucleotide.

•      The palm domain also has proofreading activity.

•      It H-bonds with the base pairs in the minor groove. It is not sequence-specific but occurs only when the nucleotides are correctly base-paired.

•      If a mismatch occurs, replication slows down and the palm domain is not able to make contact with the minor groove.

•      This frees the primer: template junction to move and make contact with the exonuclease site.

•      The exonuclease site removes the incorrect base from 3’ to 5’ direction in a process called proofreading.

•      After excision is complete, the primer: template junction slides back to the replication active site.

Watch animations on DNA Replication Overview from Pearson

Watch animations on Chemistry of DNA Synthesis from Pearson