Lecture 8 - DNA replication Flashcards
How many kb are in the E. Coli genome?
4639 kb
What is the space between each base in E. Coli?
0.34nm
What is the total length of the E. Coli genome?
1.6mm
(E. coli cell is 1-2 micro-metres)
How is bacterial DNA compacted?
By looping and supercoiling
How many Gbp is our human haploid genome?
3.2 Gbp
In the typical somatic cell, how many Gbp of DNA are there?
6.4
What is the total length of DNA in our typical somatic cell?
2.2 m
What is the diameter of the nucleus?
6 um
How is eukaryotic DNA organised?
DNA duplex - 2nm diameter
Wrapped around histones to make nucleosomes - 11nm diameter
Coiled to make chromatin fibre - 30 nm diameter
Coiled chromatin fibre - 300nm diameter
Coiled coil - 700nm diameter
Metaphase chromatid - 1400nm diameter
How often are there errors in DNA replication?
1,000,000,000,000
1 x 10^9
What did Watson and Crick predict with the mechanism of replication?
Each DNA strand can act as a template to build a complementary copy
Daughter molecules would have the same sequence as the parental molecules
Semi-conservative replication = one new and one old strand
What were the 2 predictions Watson and Crick made?
- Strands must be anti-parallel: A with T, G with C
- DNA replication should be semi-conservative: each can act as a template for DNA replication
How many hydrogen bonds are there between A and T?
2 hydrogen bonds
How many hydrogen bonds are there between G and C?
3 hydrogen bonds
What were the 3 suggested models of DNA?
- Semi-conservative: one parent and one new
- Conservative: parent conserved, daughter helix is new
- Dispersive: parent helix broken into fragments and assembled into 2 new helices
What is caesium chloride equilibrium density gradient centrifugation?
Technique that separates molecules on the basis of their densities
When was the Meselson and Stahl experiment?
1958
What was the Meselson and Stahl experiment?
- Macromolecules suspended in CsCl solution
- Solution spun forming CsCl density gradient
- Growth E.Coli in either N-14 or N-15
- Spun at 140,000 times the force of gravity
What was the Meselson and Stahl ‘most beautiful experiment?
- E. Coli grown for 14 generation on 15-N medium
- E.Coli washed and switched to 14-N medium
- Mixed with CsCl
- Solution spun at 140,000 times force of gravity forming density gradient
- Photographed under UV light and developed
What were the results from the ‘most beautiful experiment?’
Only 3 forms of DNA seen: heavy, light and hybrid
After 1 gen - all DNA hybrid
Later generations - gradual loss of hybrid in N-14/N-15 and replacement with light DNA
DNA is semi-conservative
What was the Arthur Kornberg and the discovery of DNA polymerase experiment?
Took E. Coli protein extracts
Worked out conditions required for the extract
Expectation that there would be an enzyme capable of replicating DNA
Reaction mixture treated with acid - dNTPs remain soluble but polynucleotides precipitate
Phosphorus found in pellet - new DNA present
Conclusion: new strands extended in 5’ –> 3’ direction by a polymerase
What was required by Arthur Kornberg to discover DNA polymerase?
- Template DNA
- Deoxynucleotide triphosphates
- Co-factor = Magnesium ion
- Energy source = ATP
- Primer = free piece of DNA with a free 3’ OH
What are the other acitvities of DNA polymerase?
5’-3’ DNA polymerising activity
3’-5’ exonuclease activity - mistake correcting
5’-‘3 exonuclease activity
Suggests DNA polymerase has editing/proof-reading functions and can correct mistakes
What DNA polymerase is used in the DNA replication of E.Coli?
DNA polymerase III
Lacks a 5’-3’ exonuclease activity
How are nucleotides incorporated into DNA?
2 phosphates are removed by breaking bond
Last phosphate is incorporated into the chain
What is DNA polymerase commonly envisaged as?
3 fingered hand
Conformational change (fingers tighten) only around the correct nucleotide
DNA in palm - provides environment where pyrophosphate is removed and nucleotide incorporated into strand
What did Arthur Kornberg realise?
- dNTPs are required for DNA synthesis
- DNA contains only mono-phosphate residues, so pyrophosphate is released
- Radiolabelled dNTPSs can be used to test whether DNA strands are anti-parallel
How can nucleotide incorporation be used to test DNA strand polarity
Radiolabelled sugar-proximal phosphate has 2 phosphates removed
Radiolabelled nucleotide is then incorporated into the DNA
DNA is then degraded with DNase that cuts the ester bond between the phosphate and 5’
Radiolabelled phosphate attached to nearest neighbour
How can nucleotide incorporation be used to test DNA strand polarity?
Radiolabelled sugar-proximal phosphate has 2 phosphates removed
Radiolabelled nucleotide is then incorporated into the DNA
DNA is then degraded with DNase that cuts the ester bond between the phosphate and 5’
Radiolabelled phosphate attached to nearest neighbour
Proportions of each base can only match if strands are anti-parallel
How can editing be done with DNA polymerase?
- Wrong nucleotide can be incorporated at low frequency
- Leaves unpaired 3’ -OH end that blocks further elongation by DNA polymerase
- DNA polymerase 3’ - 5’ exonuclease activity removes mismatch to leave a base-paired 3’ -OH end
DNA replication can resume
What are the different active sites in the palm/ DNA polymerase?
One for polymerisation
One for editing - has 3’-5’ exonuclease activity
What is the problem with the replication fork?
Replication requires priming - primers are extended growing in 5’-3’ direction
One strand supports continual synthesis = leading strand
One strands new priming and extension as DNA need to be built in 5’-3’ = lagging strand, replication broken into small price that must each be extended
DNA replication must be asymmetric
