Lecture 8: DNA replication [G] Flashcards
Monday 14th October
What does the fact that bacterial DNA is usually a lot larger in size than the actual bacteria tell us?
That bacterial DNA is very compacted
When did Watson and Crick propose the DNA double helix structure?
In 1953. This revolutionarised our understanding of genetics. However, their hypothesis remained theoretical until tested and confirmed by subsequent experiments.
What ways is DNA compacted?
- By looping, with loops being attached at the base in an unknown way.
- By supercoiling
Is our DNA packed more tightly than E coli’s?
Yes
Did Watson and Crick test their double helix DNA model?
No, they left it to other people to test
What is the length of an E coli cell?
1 - 2 micrometres
How long is the DNA inside E-coli?
1.6 mm
How big is our haploid genome?
3.2 Gbp
How much DNA does a typical somatic cell have?
6.4 Gbp of DNA
What is the spacing between base pairs?
0.34 nm
What is the total length of DNA in our typical somatic cells?
6.4 x 10⁹ x 0.34 x 10⁻⁹ m
~ 2.2 m
Is our DNA packed more tightly than E coli’s?
Yes
How is eukaryotic DNA organised?[compaction]
1) DNA wraps around histone proteins to form nucleosomes (~11 nm diameter).
2) Nucleosomes coil into a smaller 30 nm fiber.
3) Fiber forms higher-order loops (~300 nm diameter).
4) Loops coil further to create chromosome arms (~700 nm diameter).
5) Final condensed chromosome width: ~1400 nm.
Is the error rate for DNA replication high or low?
Low
What is the error rate for DNA replication?
~1 in 109 base pairs in most organisms
How many mutations do we generate at each cell division?
About 6 mutations
What did Watson and Crick say on April 25, 1953?
- “It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”
- Can copy DNA by making a complementary copy to each of the strands.
Are the daughter strands in DNA replication identical?
Yes
What were the 2 predictions by Watson and Crick in regards to DNA replication?
1) DNA strands are held together by ‘Watson-Crick’ base- pairing, consistent with Chargaff’s rules. To make this work, the strands must be anti-parallel.
2) Each strand is therefore complementary to the other, so each can act as a template for DNA replication. DNA replication should therefore be semi-conservative
Describe semi-conservative replication
Each daughter DNA molecule contains one parental strand and one newly-replicated strand.
WATSON-CRICK model
Describe conservative replication
The entire parental DNA molecule remains intact, and a new, separate double-stranded DNA molecule is synthesized.
Describe dispersive replication
parent helix is broken into fragments, dispersed, copied and then assembled into two new helices with interspersed old and new DNA.
Who tested models of DNA replication in 1958?
Meselson and Stahl
How did Meselson and Stahl investigate DNA replication?
Using the technique of Caesium chloride (CsCl) equilibrium density gradient centrifugation, a technique that separates molecules based on their densities.
- Take 2 strong glass tubes and add a solution of a very heavy salt, caesium chloride (CsCl).
- Spin the test tubes very fast for a long time
- This forms a CsCl gradient, where there’s a higher concentration of salt at the bottom of the tubes and vice versa.
- Take samples and weigh equal volume samples.
How did Meselson and stahl establish their conditions for the DNA replication experiment?
- They grew E coli for ~14 generations in medium containing 14N NH4Cl. (light nitrogen)
- They did the same in a medium of 15N NH4Cl. (heavy nitrogen)
- They prepared DNA from both of these tubes, mixed them together, mixed them with CsCl.
- They then made a balanced tube of the same weight and spun this at 140,000 x g
for 20hrs. - They hoped that the 2 different DNA’s would have different densities because of the weight of the nitrogen.
- Photographing under UV light allowed the different bands of DNA to be identified.
Describe the results of Meselson and Stahl’s experiment (read)
- Only three forms of DNA were seen: H, hybrid and L: after one generation, all the DNA was hybrid.
- For later generations there was a gradual loss of hybrid 14N/15N DNA and replacement with light DNA.
- Hence proving that DNA replication is semi conservative.
What led Arthur Kornberg to believe that here would be an enzyme capable of replicating DNA: a DNA polymerase?
Beadle and Tatum’s ‘one gene, one enzyme’.
How did Arthur Kornberg discover DNA polymerase?
Kornberg took Escherichia coli protein extracts, and worked out the in vitro conditions that were required for this extract to make new DNA:
1) A template DNA to copy
2) deoxyucleotide triphosphates (dATP, dTTP, dCTP and dGTP) (building blocks of DNA)
3) the co-factor Mg2+
4) an energy source, ATP
5) … and a small piece of DNA (a PRIMER) with a free 3’ OH
Kornberg’s experiment
- Primer bound to template DNA
- Added 3 non-radioactive nucleotides and a radioactive nucleotide (dTTP*).
- Added E Coli protein extract.
- After the reaction, the reaction mixture was treated with acid: unincorporated dNTPs remain soluble, but polynucleotides precipitate.
- 32P was found in the pellet – so therefore there was new DNA present.
- Radioactive labelling found that new strands are extended in the 5’ → 3’ direction by a polymerase (pol 1)
What are the functions of pol 1
- Could extend DNA strand from 5’ to 3’
- 3’ → 5’ exonuclease activity
- 5’ → 3’ exonuclease activity
- This suggests that Pol I has editing/proof-reading functions and can correct mistakes, such as incorrectly paired nucleotides.
Did Kornberg’s experiments confirm key predictions of the Watson-Crick model?
Yes:
- 1) DNA strands are held together by ‘Watson-Crick’ base- pairing, consistent with Chargaff’s rules. To make this work, the strands must be anti-parallel.
- 2) Each strand is therefore complementary to the other, so each can act as a template for DNA replication. DNA replication should therefore be semi-conservative
Pol III is essential for DNA replication in E. coli.
Pol I is NOT the enzyme that is used for replication of E. coli DNA.
What is DNA polymerise often envisaged as?
a three-fingered hand. Kornberg proposed that the polymerase enzyme works like a “hand,” where the “palm” holds the DNA and catalyzes the addition of new nucleotides. The enzyme “closes” around the incoming nucleotide to facilitate the chemical reaction.
There is a conformational change in DNA polymerase (its ‘fingers’ tighten) only around the correct nucleotide
Does polymerase edit mistakes in DNA?
Yes
is is true that Pol III lacks 5’ → 3’ exonuclease activity?
Yes
How kornberg tested strand polarity
- Starting nucleotide radioactive
- After the action of DNAse, the neighbouring nucleotide is radioactive.
- How often was nearest neighbour a T?
- GpA (0.065) = TpC (0.061)
- These proportions can only match if the strands are anti-parallel
How did kornberg test Watson-Crick base pairing?
Using a template strand with a known sequence.
Providing dNTPs with specific radioactive labels.
Allowing DNA synthesis to proceed and analyzing the composition of the newly synthesized DNA.
Observing that complementary base pairing (A-T, G-C) followed Watson-Crick rules.
He showed that the proportions of incorporated nucleotides matched the base composition of the template strand.
How does editing work with DNA polymerase?
① The wrong nucleotide can be incorporated at low frequency
② This leaves an unpaired 3’-OH end that blocks further elongation by DNA polymerase
③ DNA polymerase 3’ → 5’ exonuclease activity removes the mismatch to leave a base-paired 3’-OH end
DNA replication can now resume
What are the 2 active sites for polymerisation for?
one for polymerisation, and the other for editing. The editing site has the 3’-5’ exonuclease activity.
Is it true that new DNA is made in the 5’ → 3’ direction?
Yes
① Replication requires priming
② The primers are extended, growing in the 5’ → 3’ direction
③ Because new DNA is built 5’ → 3’ this strand needs new priming and extension … lagging strand
④ … whereas this strand supports continual synthesis (LEADING strand
)
Is it true that DNA polymerase has a proof-reading 3’ → 5’ exonuclease activity?
Yes
Who showed that DNA polymerase III is primarily responsible for large-scale DNA replication in cells?
Thomas Kornberg. ( Pol I was mainly involved in smaller, more specific repair and replication tasks.)
Are there 2 different active sites for polymerisation and for editing errors?
Yes
What did Arthur Kornberg use radioactive nucleotides to do?
To trace the incorporation of individual nucleotides into newly synthesised DNA. This allowed him to track the process of DNA synthesis
How does DNA polymerase replicate DNA on the leading strand?
In a continuous manner, towards the replication fork
How does DNA polymerase replicate DNA on the lagging strand?
In short strands, called Ozaki fragments, away from the replication fork. This requires repeated priming.
Which ion is critical to the role of DNA polymerase?
Mg2+
What did Kornberg realise was needed to initiate DNA replication?
A primer
What are the challenges faced with lagging strand synthesis?
- As the fork progresses, single-stranded DNA is exposed.
- New primers must be laid down periodically for continued synthesis.
How did Kornberg validate Watson Crick base pairing?
- Kornberg used radiolabeled nucleotides to confirm complementary pairing.
- Found consistent proportions of A-T and G-C pairing across strands.
How did Kornberg validate Watson Crick antiparallel strands?
Base pairing and nucleotide ratios validated DNA’s antiparallel orientation.
Summarise the key features of DNA replication
- Semi-conservative.
- Antiparallel in strand orientation.
- Directional: 5’ → 3’ synthesis.
- Semi-continuous: Leading strand is continuous; lagging strand is fragmented.
- Proofreading minimizes errors to ~1 in 10⁹ bases.
- Requires multiple enzymes and priming for lagging strand synthesis.
