Capture of linear fragments at a double-strand break in yeast

Abstract

Double-strand breaks (DSBs) are dangerous chromosomal lesions that must be efficiently repaired in order to avoid loss of genetic information or cell death. In all organisms studied to date, two different mechanisms are used to repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ). Previous studies have shown that during DSB repair, non-homologous exogenous DNA (also termed 'filler DNA') can be incorporated at the site of a DSB. We have created a genetic system in the yeast Saccharomyces cerevisiae to study the mechanism of fragment capture. Our yeast strains carry recognition sites for the HO endonuclease at a unique chromosomal site, and plasmids in which a LEU2 gene is flanked by HO cut sites. Upon induction of the HO endonuclease, a linear extrachromosomal fragment is generated in each cell and its incorporation at the chromosomal DSB site can be genetically monitored. Our results show that linear fragments are captured at the repaired DSB site at frequencies of 10(-6) to 10(-4) per plated cell depending on strain background and specific end sequences. The mechanism of fragment capture depends on the NHEJ machinery, but only partially on the homologous recombination proteins. More than one fragment can be used during repair, by a mechanism that relies on the annealing of small complementary sequences. We present a model to explain the basis for fragment capture.

Figure 1.

Genetic system used. The URA3::ACT1-i::HOcs is located on Chromosome V. The LEU2 gene is carried by a TRP1-marked centromeric plasmid. (A) ‘Direct’ LEU2 orientation. Upon transfer of cells to galactose, the HO endonuclease cleaves the three HO cut sites. The ‘Direct’ LEU2 fragments can be inserted in either orientation. Crossed HOcs boxes represent additional mutations required to inactivate the HOcs and prevent it from being cut again. Note that only the upper orientation allows annealing of both ends. (B) The ‘Inverted’ LEU2 fragment. Note that in both orientations, only one end can undergo simple annealing.

Figure 2.

Sequence of the repaired DSBs at the URA3::ACT1-i::HOcs locus. (A) DNA sequences of independent 5-FOA^R, Leu⁺ colonies of a wild type strain. The HOcs at the URA3 cassette is colored red, the HOcs from the linear LEU2 fragments is colored black. The new sequences at the insertions are colored blue. The insertions are shown in bold. (B) Events obtained in the rad52 genetic background. (C) Events obtained in the rad51 genetic background.

Figure 3.

Capture of linear LEU2 fragment into a DSB at the URA3::ACT1-i::2 inv HOcs locus. After HO endonuclease cleavage the termini of the broken chromosome V cannot anneal. (A) For the ‘Direct’ LEU2 fragment all samples carried two inverted HO cut sites with GT/AC insertions upstream of LEU2, and a single HOcs with a similar mutation downstream. (B) For the ‘Inverted’ LEU2 fragment all colonies analyzed carried two inverted HOcs exhibiting GT/AC insertions on one end of the fragment, and complex events on the other. (C) For the ‘Inverted fit’ LEU2 fragment, none of the events involved the use of the small inverted HOcs fragment. (D) Proposed mechanism of fragment capture (a single HOcs with a ‘Direct’ fragment are shown. The HO endonuclease cleaves both HO cut sites in the ‘Direct’ plasmid and the HOcs at the URA3 cassette, resulting in 4-bp 3′ overlapping sequences. Annealing between the terminal Adenine of the bottom strand and one T nucleotide prior to the terminal in the top strand, followed by DNA synthesis and ligation creates a GT insertion. DNA synthesis requires the removal of the terminal 3′ T nucleotide from the top strand.

Figure 3.

Capture of linear LEU2 fragment into a DSB at the URA3::ACT1-i::2 inv HOcs locus. After HO endonuclease cleavage the termini of the broken chromosome V cannot anneal. (A) For the ‘Direct’ LEU2 fragment all samples carried two inverted HO cut sites with GT/AC insertions upstream of LEU2, and a single HOcs with a similar mutation downstream. (B) For the ‘Inverted’ LEU2 fragment all colonies analyzed carried two inverted HOcs exhibiting GT/AC insertions on one end of the fragment, and complex events on the other. (C) For the ‘Inverted fit’ LEU2 fragment, none of the events involved the use of the small inverted HOcs fragment. (D) Proposed mechanism of fragment capture (a single HOcs with a ‘Direct’ fragment are shown. The HO endonuclease cleaves both HO cut sites in the ‘Direct’ plasmid and the HOcs at the URA3 cassette, resulting in 4-bp 3′ overlapping sequences. Annealing between the terminal Adenine of the bottom strand and one T nucleotide prior to the terminal in the top strand, followed by DNA synthesis and ligation creates a GT insertion. DNA synthesis requires the removal of the terminal 3′ T nucleotide from the top strand.