8. DNA replication 2 Flashcards
The mechanics of DNA replication
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DNA replication recap
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DNA replication 2
- > 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
Polymerase
-> 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
Polymerases
- > 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)
DNA polymerases
- > 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
DNA synthesis occurs in the 5’ to 3’ direction
- > 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
Problem
DNA is tightly coiled and double-stranded, so not suitable
to act as a template for synthesis!
The mechanics of DNA replication -> unwinding the DNA
- > 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
- ) Inhibitor proteins bind to replication origin
- ) DNA helicase binds to initiator protein
- ) helicases load onto DNA
=> DNA is now single stranded + can act as template for synthesis
Problem
DNA polymerase cannot start synthesis on its own, it needs a free 3’ - OH onto which to add nucleotides
Priming DNA synthesis
-> 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
- ) Helicase denatures helix + binds with DNA primase to form primosome
- ) Primase synthesises RNA primer - extended as DNA chain by DNA polymerase
Problem
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?
New templates for synthesis
- > each replication fork has an origin + direction of travel
- > AS DNA unwinds, it reveals new nucleotides to act as a template
The problem of antiparallel strands
- > 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
Continuous synthesis of the leading strand towards the replication fork
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The solution -> OKASAKI FRAGMENTS
-> 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
semi discontinuou synthesis
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Lagging strand -> Okasaki fragments
a. ) RNA primer copied from DNA
b. ) DNA polymerase III elongates RNA primers with new DNA
Okasaki fragments
- > 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
Ligation og Okasaki fragments
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
Action of DNA ligase (like molecular glue)
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
Semi discontinuous synthesis
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
Bidirection replication
- ) replication of origin sequence
- ) progression to two replication forks moving in opposite directions
=> replication can go in both directions
=> continuous in one and discontinuous in the other direction
Replisome complex
Model for ‘replication machine’, or replisome - complex of key replication proteins with DNA at replication fork
