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DNA uses Semi-Conservative Replication
DNA uses Semi-Conservative Replication
The theory that DNA replication was semi-conservative was confirmed by the Meselson-Stahl experiment in 1958
The theory that DNA replication was semi-conservative was confirmed by the Meselson-Stahl experiment in 1958
Prior to this experiment, three hypotheses had been proposed for the method of replication of DNA:
- Conservative Model – An entirely new molecule is synthesised from a DNA template (which remains unaltered)
- Semi-Conservative Model – Each new molecule consists of one newly synthesised strand and one template strand
- Dispersive Model – New molecules are made of segments of new and old DNA
Messelson and Stahl Experiment
Messelson and Stahl Experiment
Meselson and Stahl were able to experimentally test the validity of these three models using radioactive isotopes of nitrogen
- Nitrogen is a key component of DNA and can exist as a heavier 15N or a lighter 14N
DNA molecules were prepared using the heavier 15N and then induced to replicate in the presence of the lighter 14N
- DNA samples were then separated via centrifugation to determine the composition of DNA in the replicated molecules
The results after two divisions supported the semi-conservative model of DNA replication
- After one division, DNA molecules were found to contain a mix of 15N and 14N, disproving the conservative model
- After two divisions, some molecules of DNA were found to consist solely of 14N, disproving the dispersive model
The predictions of the semiconservate replication hypothesis were proved by the actual results
This video Summarizes the experiment that Meselson and Stahl did to determine how DNA replicates.
Can you explain this experiment in words for 8 marks?
Enzymes of DNA Replication
Enzymes of DNA Replication
Can you draw, label and annote the mechanisms of DNA Replication?
DNA replication is a semi-conservative process that is carried out by a complex system of enzymes
Helicase
- Helicase unwinds and separates the double-stranded DNA by breaking the hydrogen bonds between base pairs
- This occurs at specific regions (origins of replication), creating a replication fork of two strands running in antiparallel directions
DNA Gyrase
- DNA gyrase reduces the torsional strain created by the unwinding of DNA by helicase
- It does this by relaxing positive supercoils (via negative supercoiling) that would otherwise form during the unwinding of DNA
Single Stranded Binding (SSB) Proteins
- SSB proteins bind to the DNA strands after they have been separated and prevent the strands from re-annealing
- These proteins also help to prevent the single stranded DNA from being digested by nucleases
- SSB proteins will be dislodged from the strand when a new complementary strand is synthesised by DNA polymerase III
DNA Primase
- DNA primase generates a short RNA primer (~10–15 nucleotides) on each of the template strands
- The RNA primer provides an initiation point for DNA polymerase III, which can extend a nucleotide chain but not start one
DNA Polymerase III
- Free nucleotides align opposite their complementary base partners (A = T ; G = C)
- DNA pol III attaches to the 3’-end of the primer and covalently joins the free nucleotides together in a 5’ → 3’ direction
- As DNA strands are antiparallel, DNA pol III moves in opposite directions on the two strands
- On the leading strand, DNA pol III is moving towards the replication fork and can synthesise continuously
- On the lagging strand, DNA pol III is moving away from the replication fork and synthesises in pieces (Okazaki fragments)
DNA Polymerase I
- As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
- DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides
DNA Ligase
- DNA ligase joins the Okazaki fragments together to form a continuous strand
- It does this by covalently joining the sugar-phosphate backbones together with a phosphodiester bond
DNA Polymerase
DNA Polymerase
Leading and Lagging Strands
Leading and Lagging Strands
DNA polymerase cannot initiate replication, it can only add new nucleotides to an existing strand
For DNA replication to occur, an RNA primer must first be synthesised to provide an attachment point for DNA polymerase
DNA polymerase adds nucleotides to the 3’ end of a primer, extending the new chain in a 5’ → 3’ direction
- Free nucleotides exist as deoxynucleoside triphosphates (dNTPs) – they have 3 phosphate groups
- DNA polymerase cleaves the two additional phosphates and uses the energy released to form a phosphodiester bond with the 3’ end of a nucleotide chain
This explains why the leading strand is continuous and why the Lagging strand works the way it does, why an RNA Primer is necessary.
Because double-stranded DNA is antiparallel, DNA polymerase must move in opposite directions on the two strands
- On the leading strand, DNA polymerase is moving towards the replication fork and so can copy continuously
- On the lagging strand, DNA polymerase is moving away from the replication fork, meaning copying is discontinuous
- As DNA polymerase is moving away from helicase, it must constantly return to copy newly separated stretches of DNA
- This means the lagging strand is copied as a series of short fragments (Okazaki fragments), each preceded by a primer
- The primers are replaced with DNA bases and the fragments joined together by a combination of DNA pol I and DNA ligase
Very Very easy to understand, I suggest watching this one first, it explains it so clearly, really easy to understand, the other videos add a bit of complexity
Easy to understand, clear, with emphasis on the enzymes present.
Very complex video. But very clear, showing ALL the important elements of the process of DNA replication. I suggest you watch this video after the lesson, after watching the previous videos and after reviewing your notes. Use it as a summary video to check your understanding.
Structural Basis of DNA Replication
Video and Posters made by my previous students
Video and Posters made by my previous students
DNA Replication Poster Assignment
DNA Replication Poster Assignment
Try this Exam Question
Try this Exam Question
Explain the process of DNA replication. (8 marks)
Explain the process of DNA replication. (8 marks)
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