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Study Notes on Mismatch Repair (MMR) Lecture by Prof. Enni Markkanen

Mismatch Repair (MMR)

Prof. Enni Markkanen
Institute of Veterinary Pharmacology & Toxicology
University of Zürich
Email: enni.markkanen@vetpharm.uzh.ch
Bio 257 – 06.10.2025

Goals of This Lecture

The primary objectives of this lecture include:

  • Understanding and explaining:

    • How the accuracy of genome duplication is achieved

    • The role and mechanism of proofreading by DNA polymerases

    • Types of lesions subject to MMR

    • Key criteria for MMR

    • Different steps of MMR in eukaryotes and prokaryotes

    • How MMR can discriminate between new and old strands in both leading and lagging-strand contexts

    • The roles of MMR in cancer

The Problem of Accurate Duplication of the Genome

  • Size of the Human Genome: Approximately $3 imes 10^9$ base pairs

  • Replication Mechanism: Mainly performed by three DNA polymerases:

    • DNA Pol α

    • DNA Pol δ

    • DNA Pol ε

  • Error Rate of Polymerases: The error rate during replication is notably high, leading to several mistakes in each replication cycle. (Adapted from Loeb LA et al., Nat Rev Genet., 2008)

Factors Affecting the Error Rate of DNA Polymerases

The error rate of DNA polymerases is influenced by two main factors:

  1. Nucleotide Selectivity

  2. Proofreading Activity

    • These factors are contingent upon the specific polymerase, the nature of the mismatch, nucleotide pool conditions, and the local sequence context.

    • The accuracy of polymerases can improve by a factor of $10^2$ through proofreading mechanisms.

Mechanism: DNA Polymerisation vs. Proofreading

The general steps include:

  • DNA Polymerisation:

    • Involves the elongation of the newly synthesized strand in a 5' to 3' direction using a template strand.

                   5'  
Template:   ...A...  
Nascent:    ...m...  
             3'

  • Proofreading:

    • Triggered by abnormal geometries of mismatches observed by the DNA polymerase.

    • The polymerase moves backward to degrade the recently synthesized strand when a mismatched nucleotide is detected, then resumes correct sequence synthesis.

Impact of Proofreading on Tumorigenesis

  • Germline Mutations:

    • Mutations in DNA Pol ε (exo-) in mice significantly affect survival rates.

    • Study shows varied survival rates (0% to 100%) correlated with specific genotypes:

      • Genotype Groups:

      • +/+ (normal, full survival)

      • +/D400A

      • D400A/D400A (mutant, low survival)

  • Figure Analysis:

    • Age vs. survival outcome indicates the critical role of proofreading in preventing tumorgenesis.

Importance of Mismatch Repair (MMR)

  • Mismatches are unique DNA lesions with specific pairing issues, illustrated as follows:

  G:G, G:A, G:T, G:C, A:G, A:A, A:T, A:C,
  T:G, T:A, T:T, T:C, C:G, C:A, C:T, C:C

  • Mismatches consist of undamaged bases and exist only while DNA strands are annealed. MMR must act while the strands remain annealed to correct these mismatches.

  • Insertions and deletions (IDLs) result from DNA polymerase "slippage," most prominently in repetitive sequences (microsatellites).

Specific Problems with IDLs

  • Slippage in Repetitive Sequences:

    • Common in microsatellites (mono-, di-, tri-nucleotide repeats).

    • Cells deficient in MMR struggle at proofreading singular base IDLs, resulting in microsatellite instability frequently seen in MMR-deficient tumors.

   AA GCAAAA ...
   CGTTTTTTTTTTTTTTA

Proofreading vs. MMR

  • Differentiate between:

    • Proofreading: Occurs with polymerases recognizing mismatches; targets imperfectly annealed primer.

    • Mismatch Repair (MMR): Targets perfectly annealed primers requiring complex recognition systems.

Criteria for Mismatch Repair (MMR)

  1. Mismatch Recognition: MMR must detect mismatches effectively.

  2. Directed Repair: Repair mechanisms must aim to the nascent strand.

  3. Timing: Repair must occur before strand dissociation occurs.

Prokaryotic Mismatch Repair Mechanism

  • Primarily involves several proteins:

    • MutS: Recognition of the mismatch

    • MutL: Acts as a matchmaker for MMR; links MutS and repair enzymes.

    • MutH: Acts as a strand-specific endonuclease

    • UvrD: Functions as a helicase to unwind the DNA

    • Exonucleases (e.g., ExoI): Remove mismatched nucleotides

Eukaryotic Mismatch Repair Mechanism

  • The eukaryotic process employs:

    • MutSα (MSH2-MSH6): Mismatch recognition

    • MutLα (MLH1-PMS2): Molecular matchmaking and endonuclease activity

    • Involvement of PCNA and RFC in loading and repair orchestration.

MMR Functionality in Eukaryotes

  • Strand Discrimination Problem:

    • The nascent leading and lagging strands present distinct challenges in discrimination during repair processes, especially considering Okazaki fragments on the lagging strand.

  • Repair involves moving towards the gap at the beginning of these fragments, necessitating the action of subsequent proteins like Exo1 during the excision phase following mismatch detection.

Group Work Activities

Task 1: MMR and Cancer

  • Explain the implications of MMR deficiency on cancer cells.

Task 2: MMR and Microsatellite Instability

  • Discuss how MMR, microsatellite instability, and cancer are mechanistically connected.

Task 3: Methylation, DNA Damage, MMR, and Chemotherapy Resistance

  • Elucidate the mechanistic links between these concepts.

Cancer Treatment Considerations

  • Chemotherapy Agents in MMR Context:

    • Drugs: Temozolomide, streptozotocin, dacarbazine, procarbazine, etc.

    • Role of MGMT (Methylguanine methyltransferase):

      • Variability in tumor expressions of MGMT affects treatment outcomes—high expression in tumors reduces efficacy of methylating agents due to potential side effects and secondary tumors.

Final Remarks on MMR and Cancer Implications

  • Tumors deficient in MMR may not benefit from treatments using methylating agents and are likely to have adverse side effects alongside an increase in secondary tumors.