CELS191 Lecture 13: DNA Replication

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Dr. Annika Bokor

• Dr. Annika Bokor specializes in Biochemistry
• Contact Dr. Bokor via email at [email protected]
• Dr. Bokor's research interests are in Biochemistry, Genetics, and Forensics.

CELS191 2025 Lecture 13: DNA Replication

• DNA replication is the topic of Lecture 13.
• Molecular Biology and Genetics is the category of the lecture.
• Annika Bokor from the Department of Biochemistry is the lecturer.

Lecture 13 Objectives

• Explain the mechanism of DNA replication and the functions of the molecules involved.
• Explain how DNA sequence errors are corrected, and why this is important.

From Lecture 10: Genetic Material Copying

• Specific pairing in DNA suggests a copying mechanism for genetic material.
• This was noted in Nature, April 25, 1953.
• The semi-conservative model of DNA replication was introduced in Lecture 10.

Direction of DNA Synthesis

• DNA (or RNA) is always synthesised in the 5' to 3' direction.
• The 3rd carbon OH group is important to remember.
• Parental template strands are read/run in the 3' to 5' direction.

Eukaryotic DNA Replication

• Eukaryotic DNA replication involves multiple large linear chromosomes.
• Humans have 23 pairs of chromosomes.
• There are multiple origins of replication (ori).
• Replication is bidirectional.
• Parental DNA strands are teal
• Newly synthesised (daughter) DNA strands are grey.

DNA Copying Requirements

• New nucleotides (A, C, T, or G) must be progressively added.
• A starting point for nucleotide addition is needed.
• The helical double-stranded DNA must be unwound to give two parental templates.
• Tension from DNA helix unwinding must be released.
• Unwound single-stranded DNA must be prevented from reforming and protected.
• Ends of newly synthesised fragments must be joined together on both lagging and leading strands.

Semi-Discontinuous Replication

• Replication occurs in a semi-discontinuous manner.

Semi-Discontinuous Replication Defined

• The leading strand is continuously synthesised in the 5' to 3' direction.
• The lagging strand is discontinuously synthesised in the 5' to 3' direction as Okazaki fragments.
• DNA synthesis occurs toward the replication fork.

Lecture 11: RNA Synthesis Initiation

• RNA polymerase II recruits Helicase to unzip DNA by breaking H-bonds in the AT-rich promoter region.
• RNA polymerase II starts mRNA synthesis without a primer after DNA strands separate.
• RNA polymerase II has primase activity.

Primase

• Primase is an enzyme and a type of RNA polymerase that makes an RNA primer.
• Primers act as a starting point for DNA polymerisation.

DNA Polymerase III (Pol III)

• DNA Polymerase III needs an OH group for nucleotide attachment.
• It only makes DNA in the 5' to 3' direction.
• It synthesises a new DNA strand by adding nucleotides complementary to the parental template.
• It cannot bind to single-stranded DNA to initiate copying.

Initiating DNA Replication

• Topoisomerase, primase, helicase and single-strand binding proteins are involved in the initiation of DNA replication.

Making New DNA Strands

• The leading strand template leads to a leading strand being synthesised.
• Parental DNA associates with Helicase, Primer, Primase, DNA pol III and single-strand binding proteins
• The lagging strand template leads to a lagging strand being synthesised, DNA pol I and DNA ligase.

DNA Polymerase I

• DNA Polymerase I has two activities.
• It removes RNA primers (RNase H).
• It fills the gaps with DNA nucleotides (DNA polymerase).

DNA Ligase

• DNA ligase joins Okazaki fragments together.
• This creates phosphodiester bonds.

DNA Pol I Two Activities

• RNase H is an endonuclease that recognises and degrades the RNA part of DNA:RNA hybrids.
• DNA Polymerase synthesises DNA by adding complementary nucleotides to the lagging strand's parental DNA template.

DNA Ligase Role

• DNA ligase joins Okazaki fragments together to creates phosphodiester bonds.
• This occurs once RNA primers are removed and replaced by DNA nucleotides.

DNA Ligase

• DNA ligase joins lagging strand (Okazaki) fragments and newly synthesised fragments from multiple replication bubbles on leading strands.

DNA Copying Requirements Revisited

• Progressive addition of new nucleotides is done by DNA polymerase III.
• Primase enzyme makes RNA primer, creating a starting point.
• Helicase unwinds the DNA.
• Topoisomerase nicks and rejoins DNA strands, releasing tension.
• Single-stranded DNA binding protein prevents unwound DNA from degradation.
• DNA polymerase I removes RNA primers and fills gaps.
• DNA ligase joins newly synthesised fragments together.

Polymerase Chain Reaction (PCR)

• PCR is an in vitro DNA replication process done in a test tube.
• It's a lab technique used to make millions/billions of copies of a DNA section from a small original amount for detailed study.

Repairing DNA Errors

• DNA errors can be repaired DURING replication using an EXOnuclease
• DNA errors can be repaired AFTER replication using an ENDOnuclease

DNA Replication Accuracy

• DNA replication has high accuracy.
• DNA polymerase III error rate: 1 in 10^8 - 10^10 base pairs replicated.

DNA Polymerase III Proofreading

• DNA polymerase III has a proofreading mechanism.
• It checks newly inserted nucleotide bases against the template.
• Incorrect bases are removed by 3' to 5' EXOnuclease activity of DNA Pol III.

3' to 5' Exonuclease Activity

• 3' to 5' exonuclease activity of DNA polymerase III removes incorrect bases during DNA synthesis.

DNA Repair Post-Replication

• Incorrectly inserted bases not corrected by DNA polymerase III can be caused by a variety of things, for example:
• Radiation damage (e.g. UV)
• Chemical modifications of bases (natural & chemical causes)
• An ENDOnuclease removes these incorrect or damaged nucleotide bases.

DNA Errors Remediation After Replication

• Several types of DNA error and damage repair systems exist with the goal of correcting DNA errors after replication.

Importance of DNA Error Correction

• If DNA errors are uncorrected, the DNA error becomes a permanent change, damage, or mutation.

Lecture 13 Summary

• DNA replication is semi-discontinuous.
• The leading strand is synthesised continuously.
• The lagging strand is synthesised discontinuously.
• Both DNA strands serve as template strands for DNA replication.
• Eukaryotic chromosomes replicate via numerous replication bubbles that merge.
• Numerous enzymes participate in DNA replication.
• Exonucleases and endonucleases correct DNA errors during and after replication, respectively.

Objective-Based Questions

• Describe the function and importance of 7 proteins (all are enzymes) that are involved in DNA replication.
• Why does the lagging strand need to be synthesised as smaller fragments, and what are these smaller fragments called?
• Why is the primer made by the primase enzyme in DNA replication removed and replaced? (Note that the primer is not removed in transcription)
• What ‘job’ can an enzyme with a 3’ to 5’ exonuclease activity perform?
• Explain how permanent DNA errors arise.