DNA Repair.txt

  1. Base Excision Repair
    • Involves excision of single or multiple nucleotides
    • Followed by re-synthesis of correct nucleotides
    • Protects against transitions, Transversions, alkylation, deamination, etc.
  2. Components of Base Excision Repair (BER)
    • 1. DNA glycosylase
    • 2. AP endonuclease
    • 3. DNA polymerase B
    • 4. DNA ligase
  3. Base Excision Repair mechanism
    • Damage detected by glycosylase: cleaves glycosidic bond b/w sugar of nucleotide and DAMAGED base
    • AP: site of DNA lacking Base, UNSTABLE and degrades easily, yields GAP in DNA
    • AP endonuclease, DNA pol B and ligase now interact to repair
  4. Uracil DNA glycosylase (UDG) and pinch-push mechanism
    • UDG compresses DNA, through phosphate interactions with uracils
    • This causes DESTABILIZATION of the base…promoting FLIPPING of uracil out of the DNA base site
    • Residue Leu272 of UDG may be involved in PUSHING uracil out (debated)
    • Leu272 STABILIZES AP site until DNA Pol can replace base
  5. Mismatch Repair Role
    • to Correct Base Substitution Mismatches and insertion-deletion mismatches generated during replication
Recognition of mismatches
    • Mismatched DNA DESTABILIZES DNA duplex, which kink the DNA at the mismatch site
    • Involves MutS, and its Aromatic ring stack with Phe39 and H-bond with Glu41
    • Bind to mismatch via minor groove and recognize mismatch thru conserved Phe-X-Glu motif
    • MutL (and MutH) act to find mismatch and help cut it out
    • Once cut, HELICASE moves the mismatched DNA
    • DNAP III inserts correct base
    • Ligase connects it
  6. Example of mismatched recognition with thymine
    • Phenylalanine is IMPORTANT in DNA binding and MMR activity!!
    • Phe 39 of MutS approaches DNA minor groove and stacks on the unpaired T: resulting in a sharp kink
    • Phe36 stacks with the thymine by wedging through the DNA
    • Further recognition of “T” by H-bonding to glutamic acid carboxyl group
    • Then, Mismatch binding domains bind mismatch from minor groove aided by Phe-X-Glu binding motif
  7. Features that ensure MISMATCH recognition
    • Intrahelical DNA damage recog. Accounts for fact that the mispaired bases are indistinguishable from normal bases
    • Also b/c almost all DNA contacts of MutS are to the DNA phosphate-sugar backbone and sequence
  8. MutS and Mismatch repair
    • Amino (thumb) region is required for MMR
    • MutS binds to mismatch or IDL due to higher affinity – binds to phosphate-sugar backbone of DNA
    • Binds w/high affinity to G-T and single IDLs (insertion-deletion mismatches): most frequent
    • Low affinity for C-C: rarest polymerization error
    • MutS may use the DNA duplex destabilization to recognize different mismatches
  9. MutH and MutL
    • Activated by ATP, also through HEMImethylated GATC motif.
    • Once activated, now ACTIVATES by CLEAVING UNmethylated DNA strand, leaving 5’ nick on the new DNA strand (unmethylated)
    • Same strand is nicked on other side of mismatch
  10. Three modes of MMR mechanisms
    • 1. ATP dependent movement: ATP acts to move MutS-MutL complex along helix
    • 2. MutS nucleated polymerization: MutS serves as nucleation site for polymerization of 2nd protein, prob MutL
    • 3. DNA bending: MutS-MutL homologues remain bound to mismatch with activation of downstream activities by bending of DNA
Card Set
DNA Repair.txt
DNA repair