Lecture 13 Recombination

Recombination

  • Driving force of evolution through genetic shuffling.

    • Balances favorable and unfavorable genetic changes.

    • Occurs in viruses, prokaryotes, and eukaryotes.

  • Types of recombination:

    1. Homologous recombination

    2. Site specific recombination

    3. Somatic recombination

    4. Non-homologous end joining (NHEJ) – involved in DNA repair

Homologous Recombination

  • Definition: Reciprocal exchange of DNA sequences between chromosomes with the same genetic loci.

  • Occurs in:

    • Eukaryotes (both males and females):

      • Males: Spermatogenesis

      • Females: Oogenesis

    • DNA repair processes.

  • Crossing over: Occurs in meiosis and somatic cells.

  • Can occur at any location without loss or addition of bases; involves precise sequences.

  • Involves non-sister chromatids within a pair of homologous chromosomes.

Mechanisms of Homologous Recombination

  • Occurs between synapsed chromosomes.

  • Frequency of recombination is not uniform:

    • Hotspots: Short regions (1.5-2.0 kb) where recombination occurs at 100-1000x elevated rates.

  • Influencing Factors:

    • Sequence composition

    • Chromatin organization

  • Occurs during Prophase I of meiosis, correlating with specific molecular events.

Structural Aspects

  • Chiasma: Points of crossing over between homologous chromosomes.

  • Endonuclease activity: Initiates double strand breaks, followed by processes that create single-stranded overhangs leading to strand invasion.

  • Involves D-loop formation and DNA synthesis producing heteroduplex regions (one strand from each parent).

Resolving Homologous Recombination

  • Holliday Junction: Crucial intermediate formed during recombination. Requires additional DNA breaks to resolve.

    • Outcomes:

      • Splice recombinant: Recombinant genomes generated.

      • Patch recombinant: No recombinant genomes generated.

  • Heteroduplex DNA may result in mismatches requiring resolution, leading to processes like gene conversion.

Gene Conversion

  • Non-Mendelian inheritance mechanism highlighting the resolution of mismatches.

  • Example transformations (e.g., A to C, G to T).

  • Results in the loss of one allele as observed in heteroduplex regions.

Double Strand Break (DSB) Formation

  • Involves key proteins such as Spo11 in yeast, introducing breaks in the DNA.

  • Mechanism conservation across species, with mutations in Drosophila impacting meiotic recombination.

Strand Invasion Mechanism

  • Proteins involved:

    • RecA in prokaryotes and Rad51 in eukaryotes, binding to single-stranded DNA.

    • Catalyze strand invasion and exchange between ss and ds DNA.

Site-Specific Recombination

  • Definition: Recombination occurring between specific sequences.

    • Enzymes recognizing specific sequences (e.g., Cre recombinase).

    • Applications include genetic engineering using the Cre-Lox system for conditional knockouts.

  • Target sites short, generally between 14-50 bp, with notable examples being:

    • Cre-Lox (34 bp), Flippase (FLP) (34 bp), and λ Phage Integrase.

Recombination Mechanism

  • Involves integrases functioning similarly to topoisomerases during strand cleavage and rejoining.

    • Type I & II topoisomerases alter DNA strand crossing.

  • Composed of recombinases that facilitate reversibility and specific sequence binding before and after recombination.

Somatic Recombination

  • Occurs in non-germ cells and includes processes like V(D)J recombination in the immune system.

    • Specific formation and types of cells (haploid and diploid) during yeast mating type switching.

Yeast Mating Type Switching

  • Mechanism by which haploid yeast cells switch mating types, facilitated by pheromones, leading to diploid formation and subsequent meiosis.

  • Mating type information is encased within the yeast genome, specifically at the MAT locus.

  • Switching mechanism involves the HO endonuclease initiating the process, indicating directional regulation without reciprocity.

Benefits of Recombination

  • Meiotic recombination provides genetic diversity, aiding survival and adaptability.

  • Yeast can switch their mating type, providing a reproductive advantage.