Lecture10_DNA Recombination and Repair

Molecular Biology Overview

• Course: Molecular Biology BIOL-3311 DNA Recombination & Repair

• Instructor: Alireza Senejani, Ph.D.

DNA Recombination

• Homologous Recombination

  • Essential in meiosis for generating genetic diversity and in mitosis for DNA repair.

  • Involves repairing DNA breaks and managing stalled replication forks.

Introduction to Recombination

• Homologous Recombination

  • Critical process during meiosis and mitosis.

  • Facilitates chromosome segregation and integration of genetic material.

Mechanism of Double-Strand Break Repair

• Initiation of Recombination

  • Double-strand breaks (DSB) trigger homologous recombination.

  • 5' End Resection:

    • Exonuclease action creates 3′-single-stranded ends that invade a donor DNA duplex.

General Steps of Recombination

1. Double-Strand Break Occurs:

   • Resection removes sections of DNA surrounding the 5' ends.

2. Strand Invasion:

   • The 3' end from the broken molecule invades an intact, similar DNA molecule.

3. Holliday Junction Formation:

   • Cross-shaped structures connect the two DNA molecules.

4. End Resolution:

   • Specific enzymatic cuts determine crossover types, leading to either crossover or non-crossover outcomes.

   • DNA repair leads to non-crossover results, restoring the original DNA.

Homologous Recombination in Meiosis

• Chromosomes must synapse for chiasmata formation (crossing-over).

• Correlation exists between meiosis stages and molecular DNA events.

Holliday Junction Mechanics

• Resolution of Holliday Junctions:

  • RuvC enzyme cleaves junctions producing recombination intermediates.

  • Recognizes specific sequences (e.g., ATTG).

  • The resolution process in eukaryotes is not fully understood, involving multiple meiotic and mitotic proteins.

DNA Double Strand Break Repair Mechanisms

1. Recombination-Repair System (HR)

   • Operates primarily in homologous recombination.

2. Nonhomologous End-Joining System (NHEJ)

   • An alternative repair pathway.

Types of DNA Damage

• Sources of Damage:

  • Free radicals and oxidative stress.

  • Baseline daily damage varies significantly across species.

• Examples of DNA Damage

  • Single base changes (e.g., deamination of cytosine to uracil).

  • Structural distortions from UV light causing pyrimidine dimers.

DNA Repair Systems Overview

1. Direct Repair

   • Basic removal of damage, common in certain organisms (e.g., plants, E. coli).

2. Mismatch Repair

   • Distinguishes old from new DNA strands to correct mismatches.

3. Excision Repair

   • removes damaged DNA and synthesizes a replacement.

4. Tolerance Systems (Translesion Synthesis)

   • Allows replication over damaged templates.

5. Retrieval Systems (Recombination-Repair)

   • In bacteria, daughter strand damage can be repaired via recombination with an undamaged site.

Excision Repair Systems

• Nucleotide Excision Repair (NER):

  • Recognizes and repairs bulky lesions (e.g., UV-induced damage).

  • Major subpathways: TC-NER (transcription-coupled) and GG-NER (global genome repair).

• Base Excision Repair (BER):

  • Targets damaged single bases and utilizes glycosylases for repair processes.

Eukaryotic Nucleotide Excision Repair Pathways

• Global Genome Repair:

  • Repairs any damage throughout the genome.

• Transcription-Coupled Repair:

  • Prioritizes repair of actively transcribed genes.

Role of Glycosylases in DNA Repair

• Glycosylases detect and remove modified bases by flipping them out of the helix.

• Mechanisms in DNA Repair:

  • Involves incisions, excisions, synthesizes new DNA, and ligation.

Impact of Chromatin on DNA Repair

• DNA repair is modulated by chromatin structure, requiring histone modifications.

• H2A Phosphorylation:

  • Encourages recruiting repair factors in response to double-strand breaks.

Review Questions

1. Which DNA repair system involves glycosylases and lyases?

2. What are the correct sequential steps in excision repair after DNA damage occurs?