homologous recombination - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

In-vivo DNA assembly by homologous recombination in Saccharomyces cerevisiae via single-strand annealing.

This diagram illustrates how linear DNA fragments can be joinedinside the budding yeast S. cerevisiae using its natural homologous recombination machinery—a process known as “gap-repair”. Homologous recombination most likely happens though the Single Strand Annealing (SSA) mechanism.

The DNA fragments share short regions of sequence homology (about 20-30 base pairs) at their ends. Once inside the yeast cell, 5′ to 3′ exonuclease (The yellow pac man like figure) activity removes nucleotides from the ends of the DNA, generating single-stranded 3′ overhangs. These overhangs allow the homologous regions to align and anneal to each other.

The annealing step is thought to occur through alternative-end joining (alt-EJ) or single-strand annealing (SSA), two DNA repair pathways active in yeast that do not require complex recombination machinery. After annealing, any gaps are filled in by yeast DNA polymerases and the nicks are sealed by DNA ligase (The red hexagon with the glue), resulting in the formation of a continuous DNA fragment.