Homologous Recombination
Homologous recombination is a natural safety mechanism for the conservation of DNA integrity that protects chromosomes against damage to the two DNA strands, such as double-strand breaks (DSBs) or intrastrand cross-linking. DSB repair (DSBR) is one of the most investigated of all homologous recombination repair pathways (more information about DSB repair).
The recombination mechanism has been well preserved during evolution and has always been an essential factor in cell survival. As well as its maintenance role, homologous recombination underpins many other biological pathways. It is involved in the crossover process during meiosis – the process by which the alleles are rearranged – and it is essential for chromosome segregation. It is required for mating-type switching in yeast and epitope modification in many organisms. Finally, it is involved in the propagation of several mobile genetic elements such as P elements in drosophila and group I introns and inteins.
It is interesting to note that group I introns and inteins encode meganucleases, known as intron encoded endonucleases (or “homing” endonucleases), which trigger homologous recombination reactions by inducing DSBs in their target sequences.
Homologous recombination has made a significant contribution to genome engineering, as it is the basis of gene targeting and remains the safest and cleanest way to modify a genome. Its most striking characteristic is that it enables the replacement of one DNA sequence by another with a high level of precision. For example, a deficient gene can be replaced by a functional copy in situ, without any modifications being made at another site of the genome. The gene’s function can therefore be completely restored, without it being a mere compensatory reaction, as would have been the case for random insertion. It is a genuine repair.
In traditional homologous gene targeting, a homologous fragment at the locus to be modified is introduced into the cell. Even if targeted insertion into the genome can be achieved (with a selection marker placed between the adjacent homologous DNA sequences), the majority of insertion events in most organisms will occur at random sites spread throughout the entire genome. Even if some events may well result from insertion by homologous recombination on the homologous locus, these occurrences will be rare and their identification and selection will require lengthy and complex analysis.
This is not the case in a limited number of organisms and cell types in which homologous insertion is predominant: the yeast Saccharomyces cerevisiae, the moss Physcomitrella patens, the avian DT40 lymphoid cell line and some modified or mutant strains of Escherichia coli, which express a recombination protein of phage lambda.
However, with the use of MRS technology, gene targeting by homologous recombination can increase the number of targeted insertions compared with random insertions, even in mammal cells: DSB induction at a target locus leads to a high frequency of gene targeting. This frequency can reach up to several percents of cells, and is much higher than the 10-5-10-6 achieved for non-stimulated targeted integration.
