Double-Strand Break Repair (DSBR)

DSBR is one of the major roles of homologous recombination. DSBR is a major repair pathway of the cells, but mastering DSBR is also an incredibly powerful tool for the scientist. Most of Cellectis MRS© are based on DSBR.

When a DSB is made in a DNA molecule, the cell has often no other choice than to repair it or to die from chromosome loss. There are at least two major families of repair mechanism, relying on totally different molecular machineries.

First, the break can be sealed by a simple ligation. The process is often referred to as Non Homologous End Joining (NHEJ) but it has also been called illegitimate recombination or nonhomologous recombination. This religation can be perfect, simply restoring the original sequence, or imperfect, adding a few nucleotides, or removing sequences that can encompass from 1 nucleotide up to a few Kb. In mammals, the process depends on the DNA ligase IV/XRCC4 complex (Dnl4/Lif1 in the yeast S. cerevisiae) and on the DNA-dependant protein kinase complex, including DNA-PKcs, and the Ku70 and Ku80 proteins (in S. cerevisiae, there is no DNA-PKcs). NHEJ is thought to underlie random integration during transgenesis: the transforming DNA integrating via sporadic DSBs in the chromosome.

Second, DSBs can be repaired by homologous recombination. For this, the first requirement is to have homologous sequences. Depending on where such sequences can be found, there are two major homologous recombination pathways, shown in the Figure below.

If direct repeats are flanking the DSB, repair can occur by a process referred to as Single-Strand Annealing (SSA). SSA is a non-conservative recombination pathway, resulting in the loss of one repeat and all of the intervening sequences. However, if the sequences surrounding the break can find homology anywhere else in the genome, there will be a non-reciprocal transfer of genetic material from the homologous sequence, which will be used as a donor template. This process is called gene conversion.

SSA and gene conversion are distinct process in terms of mechanism, outcome, and also of genetic requirements. Gene conversion depends on a set of proteins including Rad52, Rad51 and a number of Rad51 paralogues (Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3 in mammals), Rad54, and the BRCA1 and BRCA2 genes. SSA seems to be much less demanding, and in Saccharomyces cerevisiae, it requires only the Rad52 protein.

The efficiencies of NHEJ, SSA and gene conversion vary widely, depending on the organism and cell type. Globally, homologous recombination seems to be most active in meiotic cells, in the yeast Saccharomyces cerevisiae, in the moss Physcomitrella patens, in the avian DT40 lymphoid cell line, and in a few Escherichia coli variant strains. In most mammalian and plant cells, the efficiency of random versus homologous integration of transgenes suggests that homologous recombination is very inefficient. However, this common picture needs to be tempered, if one considers that when a chromosomal DSB can be generated at the targeted locus, homologous integration can occur in up to a few percent of the cells.
A lot of Cellectis’ MRS© rely on this process, which is a process for efficiently obtaining a DSB-induced gene conversion.