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MCB 250: Homologous Recombination

Course Information

  • Vcast 43

  • Instructor: Dr. James M. Slauch, Department of Microbiology

Introduction to Homologous Recombination

  • Definition: A DNA rearrangement process where DNA is cut and rejoined.

  • Outcome: Generates new combinations of genes, known as "recombination".

  • Meaning of "Homologous": Refers to sequences that are nearly identical.

Importance of Homologous Recombination

  • DNA Repair: Critical for repairing double-strand breaks (DSBs) and other DNA lesions.

  • Meiosis in Eukaryotes: Essential for crossovers between homologous chromosomes; ensures proper chromosome segregation and genetic diversity in gametes.

  • Gene Transmission: Facilitates the transfer of genes between closely related bacterial strains.

  • Universality: Recombination occurs across all life forms, including viruses, bacteria, archaea, and eukaryotes.

Causes of Double-Strand Breaks (DSBs)

  • DSBs can occur due to various factors, including:

    • Oxidative damage

    • Radiation damage

    • Action of cellular nucleases

Mechanisms of Recombination

The Double Strand Break Model of Recombination Initiation

  • Overview: Involves several steps, starting from the double-strand break.

    • End-Processing: Creation of 3' overhangs at the break site using helicases and nucleases.

    • First Strand Invasion: Promoted by a protein called RECOMBINASE, leading to the formation of a D-loop.

    • Second Strand Invasion: Results in further interactions with the homologous strand and facilitates continuation of the process.

    • Strand Extension: Performed by DNA polymerase to synthesize new DNA strands.

Repairing a Collapsed Replication Fork

  • Collapsed replication forks can be repaired through a homologous recombination process that shares similarities with DSB repair methodologies.

Key Enzymes in Homologous Recombination (in E. coli)

  • Overview: Recombination is catalyzed by specific proteins, indicating the presence of multiple pathways.

  • Primary Pathway Components:

    • RecA: Essential for all pathways of homologous recombination.

    • RecBCD: Initiates the process by generating a 3' overhang necessary for strand invasion.

    • RuvABC: Involved in branch migration and resolution of Holliday junctions.

    • Single Strand Binding Protein, DNA Polymerase I, and Ligase: Assist in the overall process and repair mechanisms.

RecBCD Complex Mechanism

  • Function: RecBCD acts as a helicase and nuclease that binds to the DSB, unwinding and degrading DNA strands as necessary.

  • Interplay with Chi Sites:

    • When RecBCD encounters a Chi site (sequence: 5'GCTGGTGG3'), its degradation activity shifts:

    • It stops degrading the strand with the 3' end and becomes more active on the strand with the 5' end.

    • This action generates the required 3' tail that initiates recombination.

    • Chi Site Frequency:

    • Predicted frequency in the E. coli genome: 1 Chi/65 kb

    • Actual observed frequency: 1 Chi/4.5 kb

RecA Protein Functionality

  • Structure: RecA forms filaments on single-stranded DNA (ssDNA).

    • Nucleation: A slow process leading to the initial binding of RecA to ssDNA.

    • Extension: Fast process wherein RecA continues the filament growth.

    • Disassembly: Enables the dynamic formation and breakdown of the filament in response to the cell's needs.

  • Role in Recombination:

    • RecA is crucial for mediating strand invasion and is absolutely required for all homologous recombination processes.

    • It binds to ssDNA, forming a filament that searches for homologous sequences and drives strand invasion, forming duplexes.

    • Homology Requirement: RecA requires at least 50 base pairs (bp) of homology but is more efficient with longer homologies.

Holliday Junction Dynamics

Branch Migration and Resolution

  • Branch Migration: Driven by RuvAB complex, where:

    • RuvA: Recognizes and binds to the Holliday junction.

    • RuvB: Functions as an ATP-dependent helicase to facilitate migration among the junctions.

  • Resolution by RuvC:

    • RuvC cleaves the Holliday junction in a random orientation.

    • The orientation choice impacts whether a crossover or non-crossover outcome occurs.

Role of Recombination in Meiosis

  • Meiotic Process: Homologous chromosomes must pair and recombine before the first meiotic division.

  • Significance:

    1. Ensures proper alignment and segregation into daughter cells during meiosis.

    2. Generates genetic diversity by creating new arrangements of genes in the resulting gametes.

  • Initiation of Meiosis: Commences by the formation of double-strand breaks, leading to recombination events.