Recognition and repair of deaminated pyrimidines

Cytosine deamination
1. Water can attack the 4 position and release ammonia
  1. Changes cysteine to uracil
    1. Not a normal component of DNA
    2. Changes base pairing properties
    3. Uracil would rather bind adenine not guanine
      1. Mutation occurs
2. Consequences
  1. GU mismatch base repair occurs
  2. DNA polymerases moving along the DNA strand during synthesis only care if the nucleotides are bound to those in template strands
  3. During replication, one strand ends up correctly base paired (GC)
  4. Other stand ends up incorrectly base paired (AU)
  5. Another round of replication results in the insertion of a thymine (AT)
    1. Fixed mutation
  6. If this keeps recurring then cytosine levels will be reduced
  7. Progressive loss of GC in DNA
3. Uracil repair
  1. Base excision repair
  2. A cycle of replication has already occurred and adenine has been inserted
  3. Uracil-DNA glycsoylase (monofunctional) recognises uracil
    1. Uracil is cut by the N-glycosidic bond leaving an abasic site
      1. AP endonuclease cuts out the sugar leaving a gap in the DNA
        Leaves a 3' hydroxyl and 5'dRP
        End processing enzyme such as PNKP has a kinase domain which phosphorylates 5' ends and a phosphatase domain which removes phosphate from 3' end
        Must be 5' phosphate and 3' hydroxyl for DNA polymerase to work
        Gap is repaired by short patch repair
        DNA polymerase beta fills the gap with the correct base
        DNA ligase III and XRCC1 joins the gap
    2. Uracil DNA glycosylase
      1. Specific to uracil
      2. Uracil binds into pocket by hydrogen bonds
      3. Thymine can't fit in pocket as it is sterically hindered
        5' position of thymine is packed against the ring
  4. Single strand breaks are dangerous because if a replication fork were to move through the DNA and it is not anchored it would kill the cell
4. Can be reversed by cytosine reamination
  1. Corrects pro-mutagenic G:U formed by deamination
  2. Converts A:U base pairs into mutagenic A:C mispairs
Thymine
1. Why?
  1. Thymine bound to adenine is more stable than cytosine
  2. Cytosine's instability renders uracil unreliable as the base pairing partner of adenine in DNA
  3. In DNA, intentional uracil is flagged to alert the DNA it should be there and is not removed
2. How?
  1. Thymine is made the folic acid cycle
  2. At the bottom of the cycle there is a mixture of thymine and uracil incorporated into the DNA
  3. dUTPase is an enzyme only uracil can bind to
    1. Destroys dUTP and dephosphorylates it
      1. Minimises dUTP incorporation into DNA
Cancer drugs and thymine in the folic acid cycle
1. Tomudex
  1. Destroys thymine chain and leaves uracil
  2. No TTP to compete with uracil and the cell starts to incorporate lots of uracil into the DNA
    1. dUTPase cuts uracil out the DNA
      1. Lots of single strand breaks form
        The cell cannot divide and replicate due to lots of SSBs
2. Methotrexate
  1. Blocks folic acid cycle directly and starves the cell of methyl units
  2. Not enough thymine to make DNA and eventually the cells die
  3. Effective at killing rapidly replicating cells
3. dUTP/dTTP subversion
  1. PBS2 is a bacteriophage that affects bacilli
    1. Has a thymine
    2. Lots of restriction enzymes don't work as they are not expecting a thymine
    3. Has resistance to innate immune system
  2. Too much uracil is inserted into DNA and then removed resulting in lots of SSBs
  3. dTMPase converts dMTP to dT which does nothing
  4. dUMP cycle needs to be deactivated to prevent DNA degradation
    1. dCTP deaminase is introduced
      1. Takes cytosine into DNA and removes amino group
        Enough UTP is made that dUTPase is inhibited
  5. Uracil DNA glycosylase inhibitor
    1. Sits where DNA sits and replicates interaction that the DNA makes with the enzyme
      1. Stops uracil containing DNA from being degraded
4. Thymine DNA glycosylase
  1. Removes thymine when mismatched with guanine
5. Cytosine methylation
  1. Addition of methyl group makes it more likely the amine group will be lost
  2. If it recognises thymine bound with adenine it will shred the DNA unnecessarily
Structure and function in the uracil-DNA glycosylase superfamily
1. Deamination of cytosine to uracil is a major pro-mutagenic event
2. Repair by base excision repair pathway
3. Family 1 enzymes
  1. Active against uracil in ssDNA and dsDNA
  2. Recognise uracil explicitly in an extrahelical conformation via combination of protein and bound-water interactions
  3. Extrahelical recognition requires efficient process of substrate location by base sampling by hopping or gliding along DNA
  4. Specific for uracil in DNA
  5. Well understood in HSV1
  6. Shape of pocket provides selection against adenine or guanine
  7. Entry of pyrimidines such as thymine is blocked due to a side chain of a tyrosine residue
  8. Selection of bases able to enter is mediated by hydrogen bonds in pocket
  9. Amide side chain of conserved asparagine thought to differentiate between cytosine and uracil
  10. Asparagine head group makes parallel hydrogen bond interactions with uracil ring
    1. Orientation of head group is critical for selection of uracil over cytosine
4. Family 2 enzymes
  1. Mismatch specifc
  2. Recognise widowed guanine on complementary strand
  3. Broader specificity
  4. Excise uracil from mismatches with guanine
  5. Only active for GU mispairs
  6. Deep pocket provides specificity for uracil
    1. Highly conserved catalytic aspartate and histidine
  7. MUG
5. Family 3 and 4 enzymes have similar folds and common active site motifs
  1. Family 3
    1. Excise uracil from ssDNA and dsDNA in both GU and AU mispairs
    2. No activity against GT mismatches
    3. Prefers ssDNA
    4. Resembles MUG but has aspartate and glycine rather than tyrosine
    5. SMUG
  2. Family 4
    1. Glycine in final position instead of tyrosine like family 1

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Recognition and repair of deaminated pyrimidines

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