A modified single-strand annealing model best explains the joining of DNA double-strand breaks in mammalian cells and cell extracts

Abstract

The joining of DNA double-strand breaks in vivo is frequently accompanied by the loss of a few nucleotides at the junction between the interacting partners. In vitro systems mimic this loss and, on detailed analysis, have suggested two models for the mechanism of end-joining. One invokes the use of extensive homologous side-by-side alignment of the partners prior to joining, while the other proposes the use of small regions of homology located at or near the terminus of the interacting molecules. to discriminate between these two models, assays were conducted both in vitro and in vivo with specially designed substrates. In vitro, molecules with limited terminal homology were capable of joining, but analysis of the junctions suggested that the mechanism employed the limited homology available. In vivo, the substrates with no extensive homology end-joined with equal efficiency to those with extensive homology in two different topological arrangements. Taken together, these results suggest that extensive homology is not a prerequisite for efficient end-joining, but that small homologies close to the terminus are used preferentially, as predicted by the modified single-strand annealing model.

Efficient Repair of Genomic Double-Strand Breaks by Homologous Recombination between Directly Repeated Sequences in the Plant Genome

“…In plants, three different approaches unambiguously demonstrated that extrachromosomal recombination proceeds efficiently by SSA (Puchta and Hohn, 1991;Bilang et al, 1992;De Groot et al, 1992; for review, see Puchta and Meyer, 1994). Interestingly, a similar mechanism is used for NHEJ: the majority of junctions contain small patches of homologous nucleotides between reaction partners, which is best explained by the operation of a "SSA-like" mechanism (Lehman et al, 1994;Nicolas et al, 1995;Mason et al, 1996;Gorbunova and Levy, 1997;Salomon and Puchta, 1998; for review, see Gorbunova and Levy, 1999). Thus, SSA and SSA-like mechanisms might be the most prominent mode for the rejoining of broken DNA molecules in higher eukaryotes.…”

A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA

“…In addition to that, SSA may well occur at very short homology patches of only a few nucleotides. This nonhomologous variant of the SSA mechanism represents another pathway for the direct rejoining of double-strand breaks in DNA and leads to the deletion of terminal sequences (Roth et al 1985;Thacker et al 1992;Lehman et al 1994;Nicolás et al 1995). A very efficient SSA-recombination machinery has been previously shown to exist in Xenopus oocytes (Maryon & Carroll 1989, 1991a and eggs (Lehman et al 1993) which is absolutely dependent on a 5 0 -3 0 -exonuclease activity.…”

“…The underlying mechanisms are likely to involve exonucleolytic resection of DSB-termini to generate long single-stranded tails that expose small fortuitious patches of sequence homology at which single-strand annealing may occur. Processing of these partially paired structures yields junctions of the deletion type Lehman et al 1994;Nicolás et al 1995).…”

Gene Targeting and Genetic Transformation of Plants

“…Simple ligation will result in junctions with no homology. Short stretches of homology may be a result of SSA-like mechanisms [13], while longer stretches might be from an SDSA copying of ectopic chromosomal DNA into the break [14].…”