Topic 7 – Recombinal DNA Repair (Moodle)

DSE 1 RecBCD Chi 2 BCD RecA

  • Topic 7 (Chapter 13) - Recombinational DNA Repair.

  • Key terms: RecBCD, RuvABC, RecG, PriA.

  • Date: [Insert date here].

Objectives of This Topic

  • Describe and explain the various pathways of recombinational DNA repair and nonhomologous end joining (NHEJ) repair.

  • Compare mechanisms of homologous recombination repair and NHEJ repair.

Homologous Recombination and Recombinational DNA Repair

  • Major Purposes:

    1. Creation of genetic diversity via exchange of genetic information between similar DNA molecules.

    2. Recombinational DNA repair to fix single-strand breaks (SSB) and double-strand breaks (DSB).

  • Causes of DSBs:

    • DNA replication issues (e.g., encountering SSBs).

    • Meiotic recombination events.

    • Environmental factors such as UV light or gamma radiation.

    • Oxidative damage during cellular respiration.

Effects of DNA Damage at a Replication Fork

  • Possible Outcomes: a. Translesion synthesis reads through the lesions. b. Lesions stall the replisome, resulting in a halted fork. c. SSB collapse leads to DSB. d. Lesion bypass creates a single-strand gap, allowing replication to continue downstream.

Mechanisms of Recombinational DNA Repair

  • Involves multiple steps and the processing of broken DNA ends:

    1. Processing to create 3' overhangs.

    2. Recombination to form a D-loop by invading the homologous chromosome.

    3. Second strand invasion leading to double crossover.

    4. DNA polymerase uses homologous strands as templates to restore lost information.

Two Paths for Completing DSB Repair

  • DSB Repair Pathway:

    • Extension while strands are linked resulting in two Holliday intermediates.

    • Resolved by Holliday resolvases.

  • Synthesis-Dependent Strand Annealing (SDSA) Pathway:

    • Invading strands dissociate and anneal to each other before further replication and ligation.

Homologous Recombination and Holliday Junction Resolution

  • Watch related videos for visualization of processes.

  • Emphasis on complexity and significance in DNA repair mechanisms.

Repair Process for Collapsed Replication Forks

  • When a replication fork encounters a template strand break:

    • The break is converted to a DSB.

    • Triggers a recombinational repair process that requires reattachment of the broken arm.

Recombinational DSB Repair Steps at a Collapsed Replication Fork

  1. Nuclease processing creates a 3' overhang.

  2. Recombination facilitates strand invasion creating a Holliday junction.

  3. Branch migration occurs, which may create a Holliday intermediate.

  4. Resolution of the Holliday intermediate restores a viable fork.

Fork Regression In DNA Repair

  • Branch migration can trigger fork regression:

    • Backward movement allows reannealing with the parental strand.

    • Lesion is later repaired via nucleotide-excision repair (NER).

Eukaryotic Meiosis Overview

  • Stages of meiosis: Leptotene, Zygotene, Pachytene, Diplotene.

  • Role of homologous recombination in generating genetic diversity.

Mechanisms in Eukaryotic Meiotic Recombination

  • Initiated by DSBs, requiring specific histones like H2AX for detection.

Summary of Today's Objectives

  • Concepts include double-strand break repair (DSBR), translesion DNA synthesis, recombinational DNA repair during replication, meiotic recombination, etc.

Mechanisms of DSB Formation

  • S. cerevisiae Spo11 is involved in catalyzing the formation of DSBs, linking to broken strands via specific reactions.

Preparation for Recombination at DSB

  • Early complexes bind to broken ends, processing them for recombination.

Fates of DSBs in Meiosis

  • Gene conversion can result from DNA repair processes.

Recombination During Mitosis

  • Rare but can occur due to environmental stress, affecting cell cycle progression due to DSB triggers.

Repair of Mitotic DSBs

  • Mechanisms for repair involve similar processes to meiotic recombination, including the recruitment of key proteins.

S. cerevisiae Mating Type Switching

  • DSB mediated by HO nuclease allows switching between different mating types through gene conversion.

Nonhomologous End Joining (NHEJ)

  • Mechanism where DNA is repaired without conserving the original sequence, leading to potential mutations.

Steps in NHEJ

  • Involves recruitment of Ku proteins, synapsis of broken ends, followed by filling in DNA gaps and sealing nicks to complete repair.

Application of DNA Repair Systems

  • DNA repair mechanisms can be harnessed for genome editing, demonstrating their importance beyond natural repair processes.