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The __ of the primer attacks the __ of the entering nucleotide and makes an ester bond with it. One __ is released from each added nucleotide and then is __
- 3'-OH;
- phosphate;
- pyrophosphate;
- hydrolyzed to 2 phosphates
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__ are discriminated by the steric exclusion. There is a __ in the nucleotide binding pocket of the active site
- NTPs (building blocks of RNA);
- discriminator amino acid
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steric constrict:
- 2`-OH of NTPs + discriminator aa → displacement of alpha phosphate → no bond with the 3`-OH (of the extending strand)
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palm domain of DNA polymerase
- the catalytic site
- involves binding of metal ions (Mg 2+ or Zn 2+)
- monitoring base pairing of the recently added nucleotide
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palm domain: One metal ion __. The 2nd metal ion __
- reduces affinity of 3'OH for its H → making 3'-0- → attacking alpha phosphates
- coordinates with the negative charges of beta and gamma phosphates of the entered dNTP (nucleotides) and stabilizes the released pyrophosphates
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fingers domain of DNA polymerase
- important for catalysis
- Several amino acid residues of "fingers" make bonds with the incoming dNTP (nucleotides)
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If base pairing is correct, the O-helix of fingers __
moves by 40 degrees → closing the hand (enclosing dNTP) → making stacking bonds with the nucleotide → pushing it to the proximity of the metal ions of the enzyme → stimulating catalysis
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Before connecting the Okazaki fragments, the primers are removed by __ and then replaced by DNA. The last nucleotide of the primer attached to the DNA is removed by the __. A __ fills the generated gap. The last nucleotide added to the gap is joined to the next nucleotides by a phosphodiester bond generated by the enzyme __
- RNase H;
- 5' exonuclease (because RNase H cannot remove it);
- DNA polymerase;
- DNA ligase
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Hexameric ring shape DNA helicase __
opens the 2 strands from each other. There is a pore in the center of this ring that ssDNA passes through
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Each subunit of helicase has a hair pin like structure that __
binds to a phosphate and its 2 adjacent deoxyriboses
(These hair pins are arranged like a right handed staircase, each one attached to one phosphate and the 2 deoxyriboses.)
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The DNA helicase structure uses ATP to __
pull ssDNA through the pore one nucleotide after the other
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DNA helicase has a polarity (moving 5' → 3' or 3' → 5') depending on the strand the helicase is attached to. For the lagging strand, it's __
5' → 3'
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Unwinding of DNA during replication → sending turns down the DNA → making __ supercoil
positive
Supercoil must be removed otherwise the replication cannot continue.
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During replication, the supercoil is removed by __
topoisomerases
Topoisomerase I and II break one or both strands and pass the other strand (Topo I) or both strands (Topo II) and relieve the supercoil.
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Processivity is increased by the association of DNA pol with an assembly of proteins known as __
DNA sliding clamp
- Sliding clamp is ringed around the DNA and slides along the DNA without getting dissociated
- Sliding clamp also binds to DNA pol bound to the primer:template junction.
- The whole complex moves along the DNA
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If the DNA pol is detached from the DNA the clamp keeps it around, __
DNA pol reattaches and restarts the polymerization → increased processivity
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In E. coli, __ is made of 3 copies of DNA pol III and one (5 unit) sliding clamp loader.
DNA pol III holoenzyme
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The holoenzyme interacts with DNA helicase by the t proteins. After this interaction, __
the helicase activity increases by 10 fold. Helicase generates the ssDNA with the same rate as DNA polymerization
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Helicase also interacts (weakly) with the primase, __
enhancing activity of primase by 1000 fold
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All assembled proteins together at the replication fork are also known as __
replisome
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DnaB begins to increase the single-stranded region within the origin, enabling the enzymes involved in the elongation of DNA replication to attach. DNA polymerase is attached to the primer:template junction. →
- start of DNA replication. (attachment of 3 sliding clamp, fork is completed). This represent the __ of replication of E.coli
- end of the initiation phase
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steps in the initiation phase of DNA replication
- 1. binding of origin recognition complex (ORC) to the origins of replication on the DNA (happens in all phases of the cell cycle)
- 2. formation of preRC (rereplicative complex): occurs in early G1 phase. At this step, helicases are inactive and do not unwind the double strand. Helicase loading proteins are released.
- 3. Activation of helicases (occurs in S phase) → unwinding the double helix and loading of DNA replication enzymes onto the 2 single-stranded templates (completion of replisome)
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DNA replication happens once per cycle because:
- There is no formation of new preRCs during S phase
- In the S phase, only the previously made preRCs are becoming activated by the activity of regulatory proteins of the S phase
- After mitosis (early G1), new preRCs are made again, which in turn become activated in the next S phase
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As S phase is about to start, regulating proteins of the S phase phosphorylate and thereby inhibit the ORC and Cdc6 proteins, __ before the mitosis is completed.
preventing formation of new preRCs
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After mitosis, regulating proteins of the S phase are __
downregulated → Cdc6 and Cdt1 (that help to load helicase) are activated → new round of loading the helicases
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