DNA replication requires:
- a template DNA
- dNTPs (dinucleotide 3 phosphates)
- a primer
- DNA polymerase
- metal ions (Mg2+ or Zn2+)
The __ determines the order of nucleotides that should be added to the __ one after another.
- order of nucleotides in the template;
The __ of the primer attacks the __ of the entering nucleotide and makes __ bond with it. One __ is released from each added nucleotide & then is __
- phosphate alpha;
- an ester;
- hydrolyzed to 2 phosphates
How are dNTPs distinguished from NTPs during DNA synthesis?
- NTPs (building blocks of RNA) are discriminated by steric exclusion.
- There is a discriminator amino acid in the nucleotide binding pocket of the active site.
- steric constrict: 2'OH of NTPs + discriminator aa creates a displacement of the alpha phosphate, so it is unable to bind with the 3'OH (No ester bond occurs.)
DNA polymerase is similar to a __ hand griping the __
- primer:template junction
palm domain of DNA polymerase
- catalytic site
- involves binding of metal ions Mg2+ or Zn2+
- & monitoring base pairing of the recently added nucleotide
metals in DNA polymerase
- The palm domain binds Mg2+ or Zn2+
- One metal ion reduces affinity of 3'OH for its H > making 3'-O- > attacking alpha phosphates
- The 2nd metal ion coordinates with the negative charges of beta & gamma phosphates of the entered dNTP & stabilizes the released pyrophosphates
fingers domain of DNA polymerase
Several amino acid residues of "fingers" make bonds with the incoming dNTP
If the base pairing is correct, the O-helix of the fingers moves by 40 degrees, closing the hand (enclosing the dNTP), making stacking bonds with the nucleotide pushing it to the proximity of the metal ions of the enzyme. This stimulates catalysis.
Before connecting the Okazaki fragments, the primers are removed by __ & then replaced by DNA. The last nucleotide of the primer attached to the DNA strand 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
removal of primers & joining of fragments image
opens the 2 strands of DNA from each other
hexameric ring shape DNA helicase
(There is a pore in the center of this ring in which ssDNA passes through)
Each subunit of helicase has a hair-pin like structure that binds to a __ & __. These hair pins are arranged like a __ handed staircase.
- its 2 adjacent deoxyriboses;
- right (each one attached to 1 phosphate & the 2 deoxyriboses)
DNA helicase uses __to pull ssDNA through the pore one nucleotide after the other & has a polarity depending on __
- the strand that the helicase is attached to
- (5' > 3' for the lagging strand;
- 3' > 5' for the leading strand)
During replication, the __ supercoil is removed by __
Topo I & II break one or both strands & pass the other strand (I) or both strands (II) & relieve the supercoil.
Processivity is increased by __
the association of DNA polymerase with an assembly of proteins known as DNA sliding clamp.
The sliding clamp is ringed around the DNA & slides along the DNA without getting dissociated. It also binds the DNA polymerase to the primer:template junction. The whole complex moves along the DNA
DNA pol III holoenzyme structure
- in E. coli
- made of 3 copies of DNA pol III & one 5 unit sliding clamp loader.
- DNA poll III (3 copies) are attached to the sliding clamp loader by 3 flexible linker subunits (t proteins)
All assembled proteins together at the replication fork are also known as replisome.
The holoenzyme interacts with DNA helicase by the __. After this interaction, __
- t proteins;
- the helicase activity increases by 10 fold. Helicase generates the ssDNA with the same rate as DNA polymerization
Helicase also interacts weakly with the primase enhancing activity of primase by 1000 fold.
initiation of replication in E Coli
- DnaB begins to increase the single-stranded region within the origin, enabling the enzymes involved in the elongation phase of DNA replication to attach.
- DNA polymerase is attached to the primer:template junction, which starts DNA replication (attachment of 3 sliding clamp, fork is completed). This represents the end of the initiation phase of replication of E. Coli
steps of DNA replication in eukaryotes
- 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 (prereplicative complex) - occurs in early G1 phase
- 3. Activation of helicases - occurs in S phase
Describe the formation of preRC
2 helicase loading proteins (Cdc6 & Cdt1) collaborate with the ORC to load 2 of the inactive forms of DNA helicases (Mcm2-7) next to the origin (occurs in early G1 phase)
At this step helicases are inactive and do not unwind the double strand. Helicase loading proteins are released.
Describe the activation of helicases
occurs in S phase, unwinds the double helix & loads DNA replication enzymes onto the 2 single-stranded templates (completion of replisome)
In eukaryotes, DNA replications happens __ per cell cycle, because:
- There is NO formation of new preRCs during S phase.
- In the S phase, only previously made preRCs are becoming activated by the activity of regulatory proteins of S phase.
- After mitosis (early G1), new preRCs are made again which in turn become activated in the next S phase.
What happens when the S phase is about to start (in eukaryotes)?
- Regulating proteins of the S phase phosphorylate & thereby inhibit the ORC & Cdc6 proteins.
- This prevents formation of new preRCs before mitosis is completed.
What happens after mitosis (in eukaryotes)?
- G1 phase: Regulating proteins of the S phase are downregulated
- Cdc6 & Cdt1 (that help to load helicase) are activated.
- This leads to a new round of loading the helicases.
Eukaryotic DNA replication image