DNA Replication History and Models

• The lecture focus is on the history and critical experiments of DNA replication
• Includes Meselson and Stahl and, Tsuneko and Reiji Okazaki
• The 1953 Watson & Crick paper suggests a copying mechanism for genetic material due to specific base pairing

DNA Replication Models

• Semiconservative model features parental strands separating, then each strand acts as a template to synthesize a new complementary strand
• Conservative model indicates the entire parental double helix serves as a template with a daughter DNA molecule being made of two new strands
• Dispersive model involves both parental strands fragmenting, acting as templates for new DNA, and assembling into a mix of old and new parts

Meselson and Stahl Experiment

• Designed to track parental DNA strands' fate in daughter molecules
• Developed a method that could isotopically label parental DNA strands, then separate daughter strands based on density
• Isotopes are naturally occurring, some are radioactive, some contain the same number of protons and electrons but contain additional neutrons
• Nitrogen atoms (14N) in DNA bases can be replaced by (15N) by growing cells in 15NH4Cl
• E. coli can be grown for 14 generations with 15NH4Cl as the sole nitrogen source to isotopically label DNA
• Regular DNA is regarded as "light" or "normal" while the DNA with extra neutrons is "heavy."
• Gel electrophoresis won't work, density difference is key to separation
• Density is mass/volume
• CsCl is used as the salt because of its high molecular weight which enables a very dense solution, so the DNA "floats" on the surface
• During centrifugation, sedimentation and diffusion create a CsCl gradient
• Molecules migrate until they reach the point where the CsCl density matches their own
• Mid-exponential phase cells are switched from exclusively 15N medium to exclusively 14N medium
• During the first replication cycle, a 1:1 ratio of 15N to 14N occurs in the semiconservative and dispersive models
• Observing the first few replication cycles was essential, as most chains become fully light over time
• The experiment supports semi-conservative mechanism

Okazaki Experiments

• Sought to find out if DNA is synthesized continuously, discontinuously, or by some other means
• Reiji and Tseneko started their work on the directionality paradox
• Earlier experiments indicated DNA is synthesised CONTINUOUSLY
• These experiments used insensitive autoradiography, which could only detect long DNA chains
• All well-known DNA polymerases can only elongate DNA in a 5′ – 3' direction.
• There were questions whether a yet-unknown 3′ – 5' polymerase existed
• In which direction are the new DNA strands made? The study looked at, Continous, Semi-discontinous, Discontinous and Fragmented models
• Tsuneko and Reiji Okazaki developed a different method to study DNA biosynthesis
• Many of the Okazaki experiments used nucleotides containing RADIOACTIVE atoms
• Radiation: unstable atomic nuclei lose energy in the form of radiation and radiation can be detected.
• Radioactive Thymidine is incorporated into newly made DNA
• Cells are grown at 37 °C and moved to 20 °C to slow the DNA replication
• Size of DNA containing the [3H]-thymidine is determined by a sucrose density gradient
• Alkaline-sucrose gradients are like the CsCl gradients used before, Separation is on the basis of density.
• Alkaline conditions denatures DNA, releasing labelled DNA fragments so they can sediment as single-stranded DNA according to their length and thus can be separated by length
• Radioactive DNA was present in short fragments when samples after a few seconds, around 1000 bp in size, even after 30 sec and synthesis is 500-1000 nt/s
• The size of the labeled DNA was larger when exposed to [3H]-thymidine for longer (600 sec)
• Given all early fragments were small, it supported discontinuous replication model and no evidence of a continuously synthesised leading strand.
• The findings agreed that far more consistent with small fragments being joined together (discontinuous)
• As of 2019 there has been a solution to the 50-year-old Okazaki-fragment problem
• Excision repair is responsible for the apparent "fully-discontinuous" result, in a fully discontinuous model
• Aberrant base analogues such as Hypoxanthine get used for DNA
• Aberrant base analogues such as Uracil get used for DNA in dut mutants, due to the defective dUTPase
• Aberrant RNA molecules can get used for in DNA synthesis
• DNA repair pathways remove the abberrant residue, introducing a break in the leading strand!
• Another source of leading strand fragmentation!
• The alkaline conditions further the fragmentation due to baso-sensitive RNA
• Okazaki experiment was repeated with a temperature sensitive DNA ligase mutant which means without ligase, Okazaki fragments cannot be connected. Mutant also defective in DNA repair pathways, meaning incorporated RNA nucleotides are not removed and switched to a non-alkaline sedimentation gradient so incorporated RNA nucleotides are not degraded.

Lecture summary

• DNA replication is semi-conservative and semi-discontinuous!
• Labeled nucleotides and density-based sedimentation were pivotal in this research.
• Alkaline conditions Okazaki used, and DNA repair pathways resulted in leading strand fragmentation.
• The above is why results supported the fully discontinuous model.