GER lecture 8, 9, 10

• DNA polymerase:

  Family of enzymes that catalyse DNA synthesis.

  Common overall structure (palm, fingers and thumb) but a different catalytic core protein fold.

  Palm domain catalyses the transfer of phosphoryl groups – phosphoryl transfer reaction.

  Template DNA enters from the left and newly synthesised DNA exits through the right.

  Many polymerases have additional domains with 3’ → 5’ exonuclease activity for proofreading.
• DNAPs can be subdivided into classes with different features and functions – based on amino acid sequence.

  Between the classes, only carboxylate residues in the active sites are conserved (for catalytic activity).
• DNA synthesis by DNAPs:

  DNAP synthesises complementary strand to DNA template by extending the 3’ end of existing DNA.

  • Requires free 3’ OH group

  Polymerase adds deoxyribose triphosphates (dNTPs).

  Chain elongation requires nucleophilic attack by the 3’ OH group of the primer on the innermost phosphorus atom of the a-phosphate.

  A phosphodiester bond is formed and pyrophosphate (PPi) is released.

  Mg2+ is required for DNAP to function.
• DNA polymerase active site:

  Mg2+ ions are 2 closely spaced divalent metal ions.

  • Catalytic and nucleotide-binding
  • Provide scaffold for the elongation of DNA

  Catalytic metal ions lower the pKa of the O3’ of the primer terminus, making it a stronger nucleophile.

  The nucleotide-binding metal ion contacts the phosphates of the incoming dNTP and helps align the a-phosphate.

  The –ve charged side chains of the 2 acidic amino acids (Asp) are important to coordinate the 2 divalent metal ions.
• What are the consequences of a breakdown of a replication fork?

  Toxic dsDNA breaks.
• The core catalytic domains of DNAP subfamilies are unrelated.

  • Adopt different protein folds as their catalytic cores
• What is the main enzyme responsible for DNA replication in prokaryotes?

  DNA polymerase III
• What is the catalytic domain in DNAPs?

  Palm domain
• The exonuclease is a separate domain from the polymerase domain.

  • For proofreading activity
• What is the “Klenow fragment”?

  Truncated version of a DNA polymerase I (bacterial DNAP).

  Created by removing the N-terminal portion of DNAPI, leaving only the C-terminal domain which has 5' -> 3' polymerase activity and 3' -> 5' exonuclease activity.
• Why is the “Klenow fragment” often used in the lab?

  Because it lacks the 5' -> 3' exonuclease found in normal DNAP I.

  Commonly used to fill in the gaps between Okazaki fragments in the lagging strand.
• What are the functions of DNAP domains?

  Fingers help push the newly synthesised DNA out of the active site.

  Thumb helps to stabilise the interaction of the DNAP with the DNA (increases DNAP processivity).

  Palm catalyses phosphoryl transfer.
• DNAP processivity

  Thumb + sliding clamp increase processivity by stabilising the interaction between the polymerase and the DNA template and keeping the DNAP and template in close proximity in space.
• What is the sliding clamp and what does it do?

  It is a ring-shaped structure that forms a complex with/surrounds DNAP to prevent it from falling off the template (increases processivity).

  Even if the DNA polymerase falls and dissociates, the clamp still holds it in close proximity with the template and it can easily re-bind.

  When the DNAP reaches the 5’ end of the next primer, it triggers a conformational change that releases the sliding clamp (forms an Okazaki fragment).
• Reducing errors in DNA replication: Catalytic selectivity

  • Positioning of incoming dNTP leads to a conformational change from ‘open’ → ‘closed’ form of DNAP
  • Fingers and thumb move inwards
  • Allows catalytic chemistry to take place
  • Then, enzyme returns to open form to allow the binding of the next dNTP
• Sometimes, DNA replication can incorporate mismatches.

  Unlike what was suggested by Watson-Crick base pairing, it was found that DNA bases can still pair specifically without H-bonding.

  Experiment:

  Use DNA base analogues without H-bonding capability.

  • H-bond donors/acceptors removed

  Still pair efficiently by the Klenow polymerase (DNA polymerase I).

  So, it was suggested that DNA bases pair due to shape complementarity instead of H-bonding.
• If we superimpose the shapes of the DNA bases, we’ll see that there is a consensus shape to all the bases.

  The DNAP active site has evolved to fit an overall shape that can accommodate all 4 bases when correctly paired.
• Incorrect base pairing is excluded by steric clashes with the enzyme active site.

  Incorrect base pairing -> DNAP active site cannot change to closed conformation.
• Correct base pair → active site can close down.

  As fingers close in, O-helix comes down.

  Tyr aromatic rings stack with incoming base.

  • Sharing of delocalised electrons helps to stabilise the incoming base

  Lys and Arg are catalytic residues that help to stabilise the phosphates of the incoming dNTP.

  ∴ Everything is correctly lined up for incoming dNTP

  However, if there’s a mismatch, the active site can’t close properly due to steric clashes.

  The chemistry slows down.

  This is an example of reducing errors by catalytic selectivity instead of by the thermodynamics of base pairing.
• Base pairing between A-U gives the same shape as A-T, so technically it would fit into the active site.

  But discriminator amino acids prevent the 3’ OH from fitting into the DNAP active site.

  So, RNA uracils don’t get incorporated.
• There’s a danger of incorrect base pairing due to tautomerization.

  • Structural interconversion by changing 1 atom (hydrogen).

  Amino and imino tautomers exist in equilibrium and readily interconvert between each other.

  Example:

  Tautomerization of cytosine produces a different pattern of H-bond acceptors and donors.

  Can mispair and still fits into active site.
• Reducing errors in DNA replication: Enzymatic proofreading

  Tautomerization is sensed by DNA polymerase.

  Rapid tautomeric shift back to normal cytosine destroys C*-A base pairing and distorts DNA at that location.

  This distortion is sensed by DNA polymerase and activates exonuclease activity.

  Polymerisation rate slows and DNAP back steps and unwinds 3-4 bp. This conformational change allows mismatched strand to reach the 3’->5’ exonuclease site.

  Exonuclease removes some bases, but it doesn’t know which ones are actually wrong and just removes them randomly.
• Y-family translesion polymerases can bypass DNA damage due to more open catalytic site and fixed fingers (can accommodate bulky DNA lesion).

  When DNAP III reaches the damaged site, it is replaced by Pol IV/V (Y-family polymerases) to get through the damage and fix it later.

  These enzymes are considered mutagenic because they add random bases to the damaged site just to get through it.

  However, mutations are better than a broken replication fork.

  Since they have low processivity, they just fall off after they bypass the damage and normal DNAP re-binds.
• DNA pol I removes RNA primers that are used to initiate Okasaki fragments.

  RNase H (ribonuclease) removes most of the RNA primers by degrading RNA that is base paired with DNA.

  • Does not remove the RNA that is directly linked to the DNA end because RNase H can only cleave bonds between 2 RNAs
• DNA pol I 5’→3’ exonuclease activity removes RNA or DNA directly upstream of the site of DNA synthesis. So, removes the RNA-DNA linkage left behind by RNase H.

  • Can remove the entire RNA primer if RNase H is not present

  Despite interacting with a β-sliding clamp, DNA pol I has low processivity and falls off.

  Requires NAD+ as a cofactor.
• Genomic DNA synthesis occurs at a replication fork.

  DNA replication is semi-conservative – daughter DNA has 1 strand from parental DNA.

  DNA polymerase synthesises DNA in a 5’ -> 3’ direction – extends 3’ OH group.

  Since DNA strands are antiparallel, this leads to a leading and a lagging strand.

  Lagging strand has a discontinuous mode of replication – forms Okazaki fragments.
• DNA polymerases that replicate DNA cannot unwind DNA – so they need a ssDNA template.

  DNA helicase unwinds DNA using energy from ATP hydrolysis.

  • Hexameric ring-shaped enzyme that encircles ssDNA and moves in a 5’->3’ direction
  • dsDNA cannot enter ring, so strands are separated
• Unlike DNA polymerases, RNA polymerases can synthesise RNA without a primer.

  DNA primase is a specialised RNA polymerase that makes short RNA primers to allow DNA polymerase to work.

  Leading strand only needs 1 RNA primer, while lagging strand needs a primer for each Okazaki fragment.

  Primases are error-prone, but that’s okay because they get removed anyway (by DNA pol I).
• Replication in E. coli

  E. coli has a circular genome, a single origin of replication and 2 replication forks moving in opposite directions.

  Each replication fork acts independently and has its own replication machinery.
• Replisome 

  Molecular machinery that carries out DNA replication.

  There are protein-protein interactions that increase activity and lead to a more coordinated action.
• What are the enzymes in the replisome and their function?

  • Helicase: Unwinds DNA
  • Primase: Synthesises RNA primers 
  • SSB: Single stranded binding protein
  • Protects the DNA unwound by Helicase
  • Topoisomerases: change supercoiled DNA to accommodate unwinding by Helicase
  • DNAPs: Polymerases that synthesise DNA in a 5’ → 3’ direction
  • Clamp: Loader (γ) + sliding (β)
  • Stabilise interaction of DNAP + template DNA
  • Mismatch repair enzymes: Repair mismatches in DNA after replication fork
• DNA polymerase III holoenzyme

  2 or 3 core DNA polymerase III enzymes attached to holoenzyme.

  • α – polymerase
  • ε – exonuclease activity (3’ → 5’)
  • Ө – stimulates ε

  2 or 3 τ (tower) flexible linkers – allow polymerases to move around.

  1 γ complex clamp loader.

  1 sliding clamp β-subunit.

  Replicating polymerases don’t act on their own – part of a larger holoenzyme complex.
• Sliding clamp and clamp loader

  Ring-shaped structures that encircle dsDNA.

  Forms central pores, through which DNA can pass.

  There's a layer of water molecules between DNA and the protein that facilitates the sliding of the clamp through DNA.

  Pore has a diameter of 3.5nm while DNA is 2nm (enough space).
• Sliding clamps from different organisms still have similar structures.

  • Overall 6-fold symmetry
  • Similar pore diameter

  But the actual fold and # of subunits are different.
• Sliding clamp is a ring-shaped protein and E. coli DNA is circular, so how do we load the clamp?

  γ complex clamp loader opens clamp, attaches it to DNA and closes it.

  Clamp leader is a claw-shaped machine to open the sliding clamp.

  • AAA + ATPase

  ATP binding causes a conformational change to the clamp loader into the open form – has higher affinity for sliding clamp.

  • Doesn’t actually use energy from ATP hydrolysis
  • Just ATP binding stabilises the open conformation

  Protein-protein interactions changes clamp to open confirmation.
• Clamp loader + sliding clamp complex has high affinity for regions of interface between ssDNA & dsDNA.

  • In this case, ssRNA primer and dsDNA

  Clamp loader binds to this structure and places dsDNA in the pore of the sliding clamp.

  • This stimulates ATP hydrolysis – destabilises the complex

  Now we have the ADP-bound form of the clamp loader – causes conformational change that decreases the affinity to the sliding clamp.

  Loader complex and sliding clamp separate from one another and sliding clamp closes onto DNA.
• Why do leading and lagging strands need to be coupled?

  The action of leading and lagging polymerases need to be coordinated, otherwise the synthesis of the leading and lagging strands would become uncoupled.

  • Can lead to large regions of ssDNA, which can lead to toxic dsDNA breaks

  The coordinated action of the replisome links the leading and lagging polymerases.
• Mismatch repair enzymes

  If mismatches are not corrected, they are incorporated into the 2nd round of replication as a normal base pair and become a mutation.

  • No longer appears as ‘DNA damage’, but the DNA has been incorrectly replicated

  Mismatch repair enzymes fix mismatches ASAP to prevent their incorporation into DNA.
• Damage recognition MutS (E. coli)

  Homodimer of the ABC ATPase family.

  Binds DNA in a bent conformation.

  Slides back and forth looking for DNA damage.

  Looks for a change in DNA structure (bent) rather than looking for a mismatched sequence.

  When it binds mismatched DNA (flexible and bent), there’s a conformational change and MutS closes and binds to ATP, triggering another conformational change that signals downstream proteins to begin the mismatch repair process.
• E. coli mismatch proteins downstream of MutS:

  MutL: ATPase from GHKL family

  MutH: endonuclease

  MutS conformational change due to ATP binding recruits MutL and MutH.

  MutH introduces a nick on the daughter strand – which has the incorrect base.

  Exonuclease (5’ → 3’) can be loaded onto the ssDNA created by the nick.

  Exonuclease removes DNA from the nick site all the way past the DNA damage.

  DNA polymerase then extends the 3’ end to correctly fill in the gap and repair the mismatch.