Homologous recombination

In nature, homologous recombination is a DNA maintenance pathway that protects chromosomes against damage affecting both DNA strands, such as double strand breaks (DSBs) or interstrand crosslinks. DSB repair (DSBR) has been one of the most investigated homologous repair pathways (see DSBR web page).

The recombination machinery has been well conserved throughout evolution, as an essential component of cell survival. In addition to its maintenance role, homologous recombination underlies many biological pathways. It is involved in meiotic crossovers, which are responsible for the rearrangement of alleles, as well as being necessary for proper chromosome segregation. It is important for mating type switching in yeast and epitope class switching in many organisms. Finally, it is involved in the spreading of many mobile genetic elements such as P elements in Drosophila, and group I introns and inteins.

Interestingly, group I introns and inteins code for sequence-specific endonucleases, called homing endonucleases, which trigger homologous recombination events by delivering DSBs to their target sequences (see the meganuclease web page).

Homologous recombination has significantly contributed to genome engineering, because it is the basis of homologous gene targeting. Homologous gene targeting remains the cleanest and safest way to engineer a genome. Its most striking feature is that it allows the precise replacement of a sequence with another. For example, a deficient gene can be replaced with a functional copy in situ, without any modification elsewhere in the genome. Thus, the function is completely restored; it is not a mere compensatory effect, as is the case with random integration.

In classical homologous gene targeting, a fragment homologous to the locus to be modified is transfected in the cell. If one can select for insertion in the genome (with a selectable marker placed between flanking homologous DNA sequences), the majority of the insertion events in most organisms will be found at random locations all over the genome. A few events will result from the integration at the homologous locus by homologous recombination; however, one needs to screen extensively for such rare events.

This is not the case in a limited number of organisms and cell types where homologous integration is predominant: the yeast Saccharomyces cerevisae, the moss Physcomitrella patens, the avian DT40 lymphoid cell line, and a few modified or mutant Escherichia coli strains expressing recombination protein from the lambda phage or Rac prophage.

However, even in mammalian cells, homologous gene targeting can outnumber random integrations by orders of magnitudes when using MRS©: delivering a DSB in the targeted locus results in a very high frequency of homologous gene targeting. The frequency can reach a few percent of the cells, largely above the 10^-5-10^-6 found for random integration.