mutational pathways

• Base Substitution Mutation

  Nucleotide mis-insertion step (AT to GC transition) - dGTP replaces dATP, which inserts a G, which is then replicated instead of an A

  (CG to GC transition) - dCTP is replaced by dGTP, inserting a G, which is then replicated instead of C

  both steps then result in the nucleotide being improperly incorporated and repeatedly replicated
• Prokaryotic Base Excision Repair

  Single inappropriate base removed by specific DNA glycosylase

  Either a uracil or damaged base is removed

  Each glcosylase is base specific

  AP endonuclease cleaves on the 5' side of abasic nucleotide

  ligase seals the nick
• Short and Long Patch Eukary BER

  • Glycosylase recognizes the specific error
  • Ape1 creates a nick
• Deamination of 5-methyl-cytosine creates thymine
• Programmed deamination in bacteria

  regulates nucleoside and nucleotide metabolism
• Spontaneous deamination in bacteria

  triggers base excision repair, mutagenesis
• In humans, programmed deamination causes

  immunological diversity, aid in combatting viral infections, hepatitis, or retrovirus
• In humans, spontaneous deamination

  leads to diseases, such as various cancers

  specifically at 5' CpG islands
• Oxygen induced mutagenesis is repaired via Base Excision repair of 8-oxoG
• Key Enzymes of Base Excision Repair of 8-oxoG

  MutM, MutY - drive the C:G to A:T transversion mutation pathway

  MutT drives the A:T to C:G transversion mutation pathway
• MutT is a hydrolyser

  MutM - nicks DNA backbones and glycosidic links ; kicks off GO

  MutY - nicks the glycosidic linkage, but NOT the backbone

  convert pyrimidines into purines
• Mutation Pathways from 8-OxoG mispairing with A

  1. MutM, MutY drive C:G to A:T transversion
  2. Oxidize G in DNA
  3. Thymine is produced, replicated but creates unstable backbone

  If 8OxoG is on the opposite of A, MutM and MutY are unable to act
• Mutation Pathways from 8-OxoG mispairing with A

  1. MutT drives A:T - C:G transversion
  2. dGOTP - dGOMP + PPi
  3. mutT is a hydrolyase
• Global Genome Repair (E. COLI)

  1. distortion detected
  2. UvrAB and ATP bind to the impacted area
  3. UvrB works with UvrC to recognize the mismatch and nick the sides
  4. UvrD acts as a helicase that unwinds the DNA strand
  5. DNA Polymerase and Ligase begin to act and work on repairing the strand
• Transcription Coupled Repair (E. COLI)

  MEDIATED BY MFD TRANSLOCASE

  1. Happens during transcription, RNA Pol stops at transcription bubble
  2. Mfd removes RNA from transcription bubble
  3. 2 units of UvrA and 1 UvrB bind to the bubble to fix it
• UvrD plays a role in global Nucleotide Excision Repair, but no direct involvement in Transcription Coupled Repair

  UvrD is needed to remove excised DNA containing a lesion
• Human Global Genome Repair

  • searches for mismatches with specific enzymes, MFD IS NOT INVOLVED
  • HR238 and XPE mark damaged area for repair
  • ERCC1 and XPF form a cutting complex, along with XPG to nick the DNA marked for excision
  • PCNA and DNA Polymerase reconstruct strand with DNA Ligase
• Human Transcription Coupled Repair

  • removes stalled RNA Polymerase, then moves into global genome repair
  • RNA Pol stalls
  • CSA and CS8 mark the damaged/stalled area, signaling cutting via ERC11/XPF and XPG
• What is the function of Pol N in Error Free Translesion synthesis?

  Pol N serves to switch with other DNA polymerases when the fork becomes stalled.

  When Pol N takes over, it is able to bypass the lesion created by the stalling of the fork

  Pol N is able to copy "bad" DNA great, copies "good" DNA terribly
• What are the Eukaryotic MMR Pathways?

  1. EXO1 dependent - Initiates digestion of ssDNA at a break, creating nicks on the lagging strand, only able to work on dsDNA.
  2. EXO1 independent - MLH1-PMS2 strand displacement between 5' and 3' nicks that flank a mispair, acting simultaneously with FEN1 to excise the flap.
• Steps in E COLI MMR?

  1. MutS scans for mismatch in DNA, detecting bulges via bad pairs (ADP is able to tightly bind to mispairs)
  2. MutL/MutH bind to a hemmimethylated DNA sequence, showing a newly replicated strand (MutL binds with MutH to create a scanner for GATC sites only in Eukaryotes)
  3. MutH endonuclease cleaves any unmethylated DNA
  4. Exonuclease cleaves the mismatched DNA
  5. DNA Polymerase fills the gap, ligase seals
• MMR IN HUMANS?

  1. MutLa and MutSa are searching clamps for single base MMR
  2. MutSa detects a mismatch, signaling MutLa to attach
  3. MutLa creates nicks throughout the mismatched DNA
  4. Nicks in the DNA initiate EXO1 catalyzed degradation
  5. Pol gamma is able to resume synthesis