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Principles of genetics > 8. DNA replication 2 > Flashcards

8. DNA replication 2 Flashcards

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1
Q

The mechanics of DNA replication

A

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2
Q

DNA replication recap

A

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3
Q

DNA replication 2

A
  • > key enzymes + proteins and their roles
  • > mechanics of DNA replication
  • > problem of antiparrallel strands
  • > Okzaki fragments
  • > the replisome
  • > problems + solutions -> what is the problem and how has that overcome by the enzyme and other proteins involved
  • > Enzymatic action
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4
Q

Polymerase

A

-> DNA polymerase enzyme which can synthesize new DNA strand from template sequence
-> E.Coli 5 DNA polymerases but 3 mains ones are:
=> DNA polymerase I
- repair of damaged DNA + replication
- helps bring about synthesis in new forming molecule
=> DNA polymerase II
- also important in synthesising new DNA
- implicated in repair
=> DNA polymerase III
- Multisubunit enzyme responsible for de novo synthesis of DNA

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5
Q

Polymerases

A
  • > eukaryotes 5 main polymerases (alpha, beta, gamma, delta, epsylon)
  • > much overlap in action with bacterial polymerases
  • > both eukaryotic and bacterial polymerases add nucleotides one at a time at the end of 3’ end of a DNA strand (add one at a time by base pairing)
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6
Q

DNA polymerases

A
  • > cannot initiate synthesis
  • > require primer to provide a free 3’ - OH end from which they can extend
  • > primers are important component of DNA replication

=> G-C
=> A-T

=> always 5 prime to 3 prime connection

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7
Q

DNA synthesis occurs in the 5’ to 3’ direction

A
  • > refers to newly synthesised chain
    - confusing to mix descriptions of orientation of parental + daughter chains in exam questions!!!!
  • > New nucleotides are added to the 3’ hydroxyl (-OH) group of the extending DNA chain
  • > double stranded and single stranded region
  • > complementary based pairing

=> DNA polymerase is the enzyme that catalyses DNA
synthesis

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8
Q

Problem

A

DNA is tightly coiled and double-stranded, so not suitable

to act as a template for synthesis!

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9
Q

The mechanics of DNA replication -> unwinding the DNA

A
  • > TOPOISOMERASE - relaxed supercoiled DNA after binding to double stranded DNA
  • > INITIATOR PROTEIN binds to ds DNA
  • > DNA HELICASE (unwind DNA helix) binds inhibitor protein, physically unwinds DNA causing it to denature in that requires (requires energy in the form of ATP)

-> As helicase unwinds DNA single stranded binding protein (SSB) stabilizes DNA + prevents it from forming a 2 degree structure

  1. ) Inhibitor proteins bind to replication origin
  2. ) DNA helicase binds to initiator protein
  3. ) helicases load onto DNA

=> DNA is now single stranded + can act as template for synthesis

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10
Q

Problem

A

DNA polymerase cannot start synthesis on its own, it needs a free 3’ - OH onto which to add nucleotides

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11
Q

Priming DNA synthesis

A

-> PRIMASE binds to helicase + the denatured DNA
=> primase + helicase = ‘PRIMOSOME’

  • > primase activated by helicase + synthesises short RNA primer for initiation of DNA synthesis
  • > It is this RNA primer that provides free 3’ - OH group onto which DNA polymerase III can add the first nucleotide
  1. ) Helicase denatures helix + binds with DNA primase to form primosome
  2. ) Primase synthesises RNA primer - extended as DNA chain by DNA polymerase
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12
Q

Problem

A

Since DNA strands are antiparallel only one of then has a free 3’ end pointing towards the replication fork. So the other can’t be replicated, can it?

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13
Q

New templates for synthesis

A
  • > each replication fork has an origin + direction of travel
  • > AS DNA unwinds, it reveals new nucleotides to act as a template
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14
Q

The problem of antiparallel strands

A
  • > synthesis can only follow unwinding DNA on one strand
  • > LEADING STRAND
  • > nucleotides added strictly at the 3’ - OH position of the elongation strand so in any given direction only one of the two strands has a 3’ end
  • > other strand is unavailable for new synthesis
  • > LAGGING STRAND

=> gaps need to be filled

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15
Q

Continuous synthesis of the leading strand towards the replication fork

A

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16
Q

The solution -> OKASAKI FRAGMENTS

A

-> solution to problem of replicating antiparallel strands accomplished by synthesising multiple short fragments of
DNA in the normal 5’-3’ direction and joining them together later
-> These pieces are called Okasaki fragments after Reiji and Tuneko Okasaki who discovered them

17
Q

semi discontinuou synthesis

A

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18
Q

Lagging strand -> Okasaki fragments

A

a. ) RNA primer copied from DNA

b. ) DNA polymerase III elongates RNA primers with new DNA

19
Q

Okasaki fragments

A
  • > short RNA synthesised on lagging strand -> close to replication fork
  • > DNA polymerase III lengthens the primer moving away from the replication fork and displaces SSB proteins
  • > After the replication fork has moved another RNA primer is synthesized and elongated
20
Q

Ligation og Okasaki fragments

A

c. ) DNA polymerase I removes 5’ RNA at end of neighbouring fragments by 5’ - 3’ exonuclease activity + replaces with DNA
d. ) DNA ligase joins adjacent fragments

21
Q

Action of DNA ligase (like molecular glue)

A

Action of DNA ligase in sealing the gap between adjacent DNA fragments to form longer, covalent continuous chains

=> input of energy in the form ATP requires

22
Q

Semi discontinuous synthesis

A

one okasaki fragment and another and another - then need to be stuck together

Parent DNA divides into 2 single stranded parts but only partly at the Topoisomerase

-> continuous synthesis on one strand and discontinuous synthesis on the other strand

23
Q

Bidirection replication

A
  1. ) replication of origin sequence
  2. ) progression to two replication forks moving in opposite directions

=> replication can go in both directions
=> continuous in one and discontinuous in the other direction

24
Q

Replisome complex

A

Model for ‘replication machine’, or replisome - complex of key replication proteins with DNA at replication fork

25
Q

And finally …..

A

-> High fidelity DNA synthesis gives accurate copying of the genetic sequence
-> Nucleotides are added rapidly to the growing DNA strand ~850 per second in E. coli; ~60-90 per second in human cells
-> During DNA polymerisation errors occur at a rate of ~ 1 in 106 per nucleotide added
-> Proof-reading properties of DNA polymerase and mismatch repair mechanisms act to correct errors
-> Overall error rate is thus only < 1 in 109 per nucleotide
added

26
Q

Problems replicating DNA

A
  • > DNA is packaged and twisted
  • > DNA is ds
  • > DNA is sticky
  • > DNA polymerases need primer
  • > antiparallel strands
  • > need to remove RNA primers
  • > pieces need to be linked together
  • > speed and movement

Principles of genetics (11 decks)

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