MT1: DNA replication

Polymerases, chromosona; organisation of replication and practical uses.

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  • Created by: ava.scott
  • Created on: 12-12-15 15:30

Replication Forks and DNA polymerase

The helix splots, and replication machinery moves bidirectionally, left and right of the replication origin, across each fork.

DNA polymerase requires

  • single-stranded DNA template
  • dNTP building blocks
  • RNA or DNA primers
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Replication Origins

The helix is usually very stable, due to hyrdrogen bonds between base pairs. Boiling temperatures must be reached before these break. For replication to occur the strands must separate.

  • Initiator proteins bind to specific DNA sequences called replication origins.
  • They break a single hydrogen bond, and then unzip the helix.

Prokaryotes (and yeast)

  • replication origins span around 100 nucleotides.
  • AT rich regions are easier to pull apart than GC (as a AT base pair has only 2 hydrogen bonds)
  • Circular DNA has one replication origin

Eukaryotes

  • Much larger genome, so many replication origins, with avg. 220 per chromosome.
  • Replication begins simultaneously at all these replication points

Proteins collect at the newly opened helix, and create the replication machinery.

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Polymerase action

5' to 3' direction

  • Phosphodiestser bond is formed between the 3' end of the growing chain and the 5' end pof the incoming nucelotide.
  • Nucleophilic attack of 3'OH of primer on alpha-phospahte of the incoming nucleotide.

This presents a problem, as one template strand runs in the alternative direction, and must be transcribed in the 3' to 5 ' direction. This is solved by Okazaki fragments.

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Okazaki fragments

DNA polymerase can only polymerise nucleotides in the 5' to 3' direction.

The replu=ication fork is therefore assymetric, and provides a problem.

This is solved by the backstitching of Okazaki fragments, which are small DNA fragments, made by DNA polymerase, and then ligated into the growing strand. This is the lagging strand, and is synthesised discontonulously behind the leading strand.

  • RNA primers allow small fragments to attach to the strand.
  • RNA-se removes the primers
  • DNA polymerase I extend okazaki fragement
  • dna ligase joins it to the next fragment
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Characteristics of DNA replications

As DNA helicase promotes the prying apart of the helix, using energy from ATP hydrolysis.

Single stranded dna-bdining proteins prevent the helix from reforming, and so the templates remain elongated and suitable templates.

  • PROBLEM: on the otehr side of the DNA replication fork, the helix is getting wound tigher, due to the pressure of DNA helicase.
  • Solution: DNA topoisomerases make transient nicks in the helix, releasing the tension, and allow DNA helicase to move forward.

Sliding clamp

  • keeps the DNA polymerase attached to the template strand, and so allows long continous transcription.
  • It sits on the newly formed helix, gripping the polymerase.
  • This action requires the clamp loader protein to hydrolyse ATP.
  • This occurs many times on the lagging strand, for each Okazaki fragment.
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DNA polymerase

Catalyses replication

  • Has a very low error rate woth only one mistake every 10^9 nucleotides
  • Very fast acting, replicating the huge genome many times in a few hours.

WORKS by hybridisation to each strand within the double helix.

  • These strands are exactly complementary so can serve as templates.
  • 5' to 3' polymerisation reaction involves nucleophilic attack of 3'OH of primer on a alpha phosphate of incoming nucleotide; pyrophosphate (ppi) is expelled

requirements for synthesis:

  • single stranded DNA template
  • dNTP building blocks
  • primer- RNA or DNA
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Telomerase

The lagging strand at the end of a chromsome cannot be repaired, as the primer sequence will have been removed from the previous round.

Prokaryotes solve thid by aving circular DNA, and Eurkaryotes have telomeres,which are long repeating sequences at the end of the chromosome.

Telomerase contains a specific RNA sequence within its structure, that allows the enzyme to extend the telomere by adding many copies of this repeated sequence. Theerefore, the strand will not get successively shorter over time.

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isolation of DNA polymerase

  • 100kg of E.coli (grows fst and can be harvested in large quantities)
  • obtain a cell-free extract which is separated in fractions by biochemical methods
  • identify polymerase using TTP incorporation assay
  • makes 1g of DNA polymerase I
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Discovery of DNA polymerase I

A.Kornberg assumed:

That deoxyribosenucleoside 5'triphosphtaes (dNTP's) are atcivated recursors for DNA synthesis

  • high energy bonds in phosphate groups

development of a sensitive method to detect polymerase activity (biochemical assay

  • radioactive dNTP's are soluble before exposure to DNA polymerase, upon which they make a insoluble acid ppt
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Many DNA polymerases

Both eukaryotic and prokaryotic cells have many DNA polymerases

E.COLI

  • E.coli mutants adefective in DNA polymerase I are still viable (though defective in repair)
  • E.coli has 5 DNA polymerases which function mostly in DNA repair
  • DNA polymerase III is essential and carries out chromosomal DNA replication.

Humans

  • Human cells have 16 polymerases;
  • 3 polymerases (α, δ, ε) are needed for chromosome replication.
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Autoradiograph

To depict circular E.coli chromosome

  • Grow E.coli in 3H-thymidine
  • Lyse cells and expose to x-ray film (FOR 2 MONTHS)
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Practical uses of DNA polymerases

DNA sequencing

  • dtermining the [recise order of nucelotides within a DNA moelcule, or a whole genome.

Polymerase Chain Reaction

  • starting with a minute quantity of DNA, allows amplification of specific gene from total genome
  • uses heat-resistant Taq polymerase from thermus aquaticus

Method

  • denaturation at 95 celcius, separates strands
  • primers attach at 55 celcius
  • taq polymerase works at 72 celcius
  • repeat 20-30 cycles
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Self Correction and proofreading

Polymerase senses incorrect nucleotides in the sequence, and repairs them. This is achieved by 3' to 5' econuclease activity.

  • Error rate without proofreading 1 in 10^5
  • with proofreading, 1 in 10^7
  • with post-replication repair 1 in 10^9

Ring shaped proteins (e.f. Beta-subunit clamp) hold processive DNA polymerases onto the template DNA. They sit behind the DNA, in rleation to teh direction of replication.

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