DNA Replication: PHA114 Lecture

DNA Replication Overview

• DNA replication is a molecular biology topic generally focused on in lectures during PHA114.
• A recommended biochemistry textbook by T.A. Brown is available online and at the Murray Library.

Learning Outcomes

• Understand biochemical processes in prokaryotic and eukaryotic DNA replication.
• Recognize experiments used to understand DNA replication.
• Know how DNA replication is controlled within the cell cycle.

DNA Replication Basics

• DNA replication involves a complex mechanism relating to the copying of genetic material.
• Watson and Crick's paper proposed the specific pairing of DNA suggests a copying mechanism.
• The reason DNA is a helix is just chemistry.

Hersey Chase Experiment

• Conducted in 1952 by Alfred Hershey and Martha Chase to prove DNA is the genetic material.
• Viruses grown in radioactive sulphur (35S) had radiolabelled proteins.
• Viruses grown in radioactive phosphorus (32P) had radiolabelled DNA.
• Viruses infect bacterium (E. coli), separated via centrifugation. Larger bacteria = solid pellet, smaller viruses in supernatant.
• Bacterial pellet radioactive when infected by 32P-viruses (DNA), not 35S-viruses (protein).
• Concluded, DNA, not protein, is genetic material because DNA transferred to bacteria.

DNA Replication Models

• Semiconservative replication gives two DNA molecules, each with one old and one new strand.
• Conservative replication gives two duplexes: one with two old strands, one with two new.
• Dispersive replication gives daughter duplexes as a mix of old and new strands.
• Meselson and Stahl proved semiconservative replication in 1958.

Meselson and Stahl Experiments (1958)

• Experiment used a centrifuge to separate DNA molecules labeled with isotopes of different densities.
• The experiment supported the semiconservative model of DNA replication.

DNA Replication Initiation

• The process of DNA replication is broadly similar in prokaryotes and eukaryotes.
• Eukaryotic DNA replication is more complex.
• Replication starts at an origin of replication, resulting in two replication forks.
• The eukaryote genome is much larger than the prokaryote genome.
• Eukaryotic replication involves multiple origins.

DNA Replication Mechanism

• A replication fork is a branch point in a replication eye where DNA synthesis occurs.
• Replication bubbles may have one or two replication forks (uni- or bidirectional).
• DNA replication is almost always bidirectional.
• Prokaryotic and bacteriophage DNAs have one replication origin.
• Reniji Okazaki elucidated the semidiscontinuous model of DNA replication.

Replication initiation in Prokaryotes

• Four copies of a 9-bp sequence bind DnaA proteins.
• Once all binding sites are occupied, more DnaA proteins are attracted.
• Local AT-rich region of the DNA opens up via torsional stress.
• A pair of replication forks are generated.
• DnaB is recruited to replication fork to initiate the formation of the pre-priming complex.
• DnaB = Helicase enzyme, which breaks base-pairs (hydrogen bonds).
• Open DNA strands get covered with single stranded binding proteins (SSBs).
• SSBs stop strands re-annealing and protect DNA from attack by free radicals and nuclease enzymes.
• Initiation of DNA replication is now complete, elongation is started.

DNA Polymerisation

• New strand synthesis is carried out by DNA-dependent DNA polymerase enzymes.
• DNA is generated in the 5' to 3' direction.
• The polymerase moves along the template strand in the 3' to 5' direction.

Polymerisation mechanism

• The 3’ end of the new strand favorable energetics.
• Polymerisation mechanism unfavourable energetics.

Prokaryotic elongation

• Primase enzyme (DnaG) binds near helicase, synthesises RNA primer on leading strand.
• Single strand binding proteins stabilise the lagging strand.
• DNA polymerase III holoenzyme clamps to leading strand and synthesises DNA.
• DNA polymerase III holoenzyme is a multi-subunit complex consisting of 17 polypeptides.
• Consists of four subassemblies - α, ε, θ.
• Sliding clamp comprises two homodimers of the β subunit (ring structure).
• There is an RNA polymerase enzyme associated with the core enzyme.

Semi-discontinuous replication

• DNA synthesis is carried out by DNA polymerase in the 5' to 3'️ direction.
• DNA polymerase inserts the 5'️ nucleotide first and extends towards the 3'️ end.
• The template DNA molecule is always used in the 3'️ to 5'️ direction.
• The lagging strand gets generated via the synthesis of multiple Okazaki Fragments.
• Lagging strand generated in opposite direction to the replication fork's movement.

Okazaki fragments

• DNA replication starts with a short RNA primer that allows proofreading.
• The primase enzyme does this in E. coli.
• Exonuclease activity of the polymerase complex removes the primer.
• Two DNA polymerase enzymes are tethered together to replicate both strands at the same time.
• Replication happens continuously in the 5' to 3' direction (leading strand).
• Occurs discontinuously in the 5' to 3' direction on lagging strand.
• The replisome is the combination of all the replication proteins.

Dealing with the Okazaki fragments

• The lagging strand gets looped over the top of the replisome so that both polymerases move in the same direction.
• DNA polymerase III replaced by DNA polymerase I, which removes RNA primer.
• Topoisomerase enzymes proceed ahead of the replisome.
• Ligase enzyme joins the Okazaki fragments together (ligation).

DNA Replication - Elongation

• Replication forks only progress a short distance without causing a topological problem.
• Double helix requires rotation to stop over-winding ahead of replication fork.
• Topoisomerases are specialised enzymes that alleviate problems.
  • Type I: introduces a break in one strand, passes the other through, then reseals.
  • Type II: breaks both strands, passes a double helix through the gap, then reseals.
• Enzymes covalently attach to the breaks in DNA.

Overcoming the topological problems of DNA replication

• During replication, double helix is unwound by DNA topoisomerases.
• The replication fork is therefore able to proceed without helix rotation.

Termination of the replication process

• The two replicons in E. coli meet 180°away from the origin of replication.
• Regulatory mechanism ensures replicon meet at a specific point.
• First one at the meeting point waits for the other before signaling that DNA replication is complete.
• Specific terminator sequences signal that replicon is approaching stop sequence.
• If replicon meets a transcription bubble (mRNA synthesis), it waits and doesn't overtake.
• Topoisomerase unlinks the interlinked “daughter” DNA molecules.
• Separated DNA molecules are segregated awaiting cell division. Each cell will receive a DNA molecule during cell division.

Eukaryotic DNA replication

• Eukaryotic DNA replication includes similarities with prokaryotic replication but also differences.
• Eukaryotic replication forks slower because of the complexity of eukaryotic DNA structures.
• Eukaryotic replication forks move at around 50 bp per second.
• Eukaryotic genomes need multiple replication forks: 50,000 – 100,000 per mammalian cell.

Eukaryotic DNA replication characteristics

• Clusters of 20-50 replicons initiate simultaneously in S-phase based on initiation factors access.
  • Early S-phase clusters are euchromatin.
  • Late S-phase clusters are heterochromatin.
  • Centromeric and telomeric is replicated last.
• The minimum length of a DNA molecule that will support replication is 11 bp with the sequence: [A/T]TTTAT[A/G]TTT[A/T] - additional copies are required for optimal replication efficiency.
• The sequence is bound by the Origin Replication Complex (ORC), activated by cyclin dependent kinases (CDKs).
• This simplifies the DNA duplex opening.
• It is believed replication in mammals may randomly initiate at areas of repetitive DNA sequence.

Eukaryotic DNA replication & mutations

• Eukaryotic DNA replicons can only initiate once per cell.
• This assures the DNA fully and controllably replicated prior to cell division.
• This limits the introduction of mutations.
• Protein licensing factor complex needed for initiation of DNA replication and inactivated after initial use.
• Only able to gain access to nucleus when the nuclear envelope dissolves in mitosis.
• The eukaryotic origin is identified (binding of ORC), set-up (binding of initiation factors), checked, and then replication is initiated.

Telomeres in Eukaryotic DNA replication

• Replication of chromosome ends (telomeres) cannot be done by semi-discontinuous replication.
• No DNA to elongate once the RNA primer is removed from the 5'-end of the lagging strand.
• This could potentially lead to the loss of genetic material, which would impact genetic material.
• To solve this, eukaryotic chromosomes have hundreds of non-coding sequence repeats: TTAGGG.
• The 3'-end overhangs the 5'-end.
• Telomerase associates with short RNA molecules partially complementary to this sequence.
• RNA acts as a template for the repeats addition to 3'-end overhangs.
• The complementary strand is synthesized by normal lagging strand synthesis.

Overview conclusion

• The following was been covered to help with your study:
  • Basic processes in prokaryotes and eukaryotes (and a simplified mechanism in eukaryotes).
  • Semi-conservative replication.
  • Semi-discontinuous replication.
  • The need for DNA synthesis in the 5' to 3' direction.