DSB repair by a single-strand oligonucleotide occurs via SSA.
Previously, we demonstrated that a ssDNA oligonucleotide can repair a DSB induced in yeast by the site-specific I-SceI endonuclease or a spontaneous DSB generated at the site of an IR composed of human Alu
). In the wild-type yeast strain, the frequencies of oligonucleotide-targeting events after DSB induction exceeded the frequencies of transformation in cells without a DSB ~5,000-fold for the I-SceI-induced break and 30-fold for the IR-induced break (37
) (data not shown). Therefore, the vast majority of transformation events corresponded to the repair of a DSB by single-strand oligonucleotides. Here we investigated the genetic requirements in order to identify the processes involved in the repair of these two different types of DSBs by ssDNA.
For this purpose, we constructed the haploid Saccharomyces cerevisiae
strain FRO-1 in which both types of DSBs could be generated. This strain contains the self-generating DSB cassette “CORE-I-SceI” (37
) composed of I-SceI endonuclease under the inducible GAL1
promoter, along with the I-SceI target scission site, integrated into the TRP5
locus (Fig. ). As shown in Fig. , 26 to 38% of the DNA is cut within 4 h of induction in synthetic complete medium containing 2% galactose. The FRO-1 strain also contains the previously described Alu
), which is located in the middle of the LYS2
gene. In strains of the same background, this IR leads to a spontaneous, hairpin-capped DSB in ~2% of the cells (22
) (Fig. ). A series of isogenic strains with deletions in DNA repair and recombination genes was also developed from FRO-1.
FIG. 1. Systems for generating an inducible and a spontaneous chromosomal DSB. (A) Diagram of the I-SceI-inducible DSB system, presenting the scheme of the CORE-I-SceI cassette inserted into the middle of the TRP5 gene on chromosome VII. The position of the DSB (more ...)
Following galactose induction, the various strains were transformed with single-strand oligonucleotides (Table ) designed to repair either the I-SceI break (e
) or the spontaneous IR break (w
), as described for Fig. . Cells were selected either for loss of the CORE-I-SceI cassette (Trp+
) or for deletion of the Alu
). The repair of both kinds of DSBs by single-strand oligonucleotides required the Rad52 protein (Table ) (also see reference 37
for details of the repair of the I-SceI break). The deletion of RAD59
, a RAD52
homolog, also greatly reduced repair. However, a small number of repair events (<0.1%) occurred independently from RAD52
Impact of genes involved in single-strand annealing and strand invasion on DSB repair by single-strand oligonucleotides
Rad52 is essential for both strand invasion and strand annealing (44
) and is implicated in end joining between DSBs with complementary single-strand ends longer than eight bases (9
). There was no detectable effect on DSB repair of mutations in the NHEJ genes YKU70
(Table ), consistent with the view that nearly all single-strand oligonucleotide repair of a DSB is mediated through Rad52-dependent homologous recombination. Therefore, we investigated the role of genes specific for the genetic control of strand invasion. The Rad51 protein, like RecA of E. coli
, is the recombinase that mediates strand invasion by ssDNA. The deletion of RAD51
as well as other genes involved in this mechanism (RAD55
, and RDH54
) did not reduce repair efficiency. In fact, targeting was increased as much as fivefold (Table ), which is similar to what has been observed for direct repeat recombination (25
). This increase appears to be due to sister chromatid competition for DSB repair (see below). The strong reduction in repair of the I-SceI break observed in rad52
mutants and the increased frequency of DSB repair observed in the rad51
strain were not attributable to differences in DSB formation (Fig. ). Survival after transformation was ~30 to 40% with or without oligonucleotides for the wild type and was not changed in any mutant tested.
Impact of genes involved in NHEJ and processing of recombination intermediates on DSB repair by single-strand oligonucleotides
To further address the role of SSA versus strand invasion, rad52
mutants with C-terminal truncations were examined. These mutants retain single-strand annealing function but are unable to recruit Rad51 to ssDNA (16
). Both mutants were highly sensitive to gamma radiation in manners similar to that of a rad52
-null strain, while their sensitivities to methyl methane sulfonate were intermediate between the wild-type and rad52-
null strains (not shown) in manners similar to those of other C-terminal deletion mutants of RAD52
). As shown in Table , the efficiency of DSB repair by ssDNA in the rad52
mutants was increased to levels comparable to those found in mutants of the Rad51 strand invasion mechanism. Thus, the primary pathway for single-strand oligonucleotide repair of a DSB occurs via the SSA function of Rad52.
Since large DNA regions must be lost in order to generate the Trp+
recombinants (Fig. ), we examined the roles of the RAD1
, and MSH3
genes, which normally cleave the nonhomologous tails formed during HR (for details, see reference 29
) (Table ). While the impact was much smaller than that for the rad52
mutants, the Δrad1
mutants reduced the repair of I-SceI and IR breaks by single-strand oligonucleotides up to fivefold. The Δmsh2
mutants affected only repair of the IR break. Partial dependence on Rad1/Rad10 and Msh2/Msh3 clippase is in agreement with the existence of alternative pathways for 3′ tail removal indicated for short (fewer than 30 nucleotides) 3′ tails or in situations with only one nonhomologous 3′ tail (7
). We note that in our experimental systems, only one long nonhomologous 3′ tail is formed per annealing of the oligonucleotide to the end of a DSB, and the nonhomologous tail on the other side can be very short (just 9 nucleotides of the I-SceI site).
Because of the large impact of the yeast RAD52
gene, we investigated whether the SSA function of the human Rad52 protein could also be characterized in our system. Amino acid homology between S. cerevisiae
Rad52 and other eukaryote homologs in the N-terminal regions (27
) suggests functional similarity. Since the human N-terminal domain of Rad52 corresponding to amino acids 1 to 209 can promote single-strand annealing in vitro (35
), we asked if these same 209 residues of human Rad52 isoform α could enable oligonucleotide repair of a DSB in yeast. This sequence was placed under the GAL1
promoter on an episomal plasmid containing the nourseothricin selectable marker (12
) in the Δrad52
mutant. Galactose induction results in both the expression of the human protein and the generation of a DSB at the I-SceI site within the TRP5
gene. After transformation with single-strand oligonucleotides, cells were plated to YPGal medium containing nourseothricin in order to maintain the expression of the rad52
allele and selection for the plasmid. Colonies formed on these plates were replica plated to Trp−
plates to select for the oligonucleotide repair events.
As shown in Table , the overexpression of the human Rad52 N-terminal domain resulted in significant albeit limited levels of repair by the single-strand oligonucleotides. Moreover, we show that such repair mediated by the human Rad52 N-terminal domain is similar to repair mediated by the truncated yeast protein in that it is independent of Rad51 and partially dependent on Rad59. Our results with the human Rad52 isoform α containing the N-terminal 209 residues provide the first direct evidence for human Rad52 SSA activity in vivo. We conclude that the oligonucleotide DSB repair system we developed can serve as a sensitive genetic assay for detecting single-strand annealing capacity that can be extended to a variety of Rad52 homologs with potential SSA function.
The N-terminal domain of human Rad52 mediates DSB repair of single-strand oligonucleotides in yeast
The Rad52 SSA oligonucleotide pathway for DSB repair is an efficient alternative to repair via strand invasion.
The enhanced DSB repair by SSA with oligonucleotides in the rad51
strand invasion mechanism mutants (Table ) demonstrates that this mechanism involving simple annealing of short stretches of complementary DNAs can be highly efficient. Since only 30% of molecules have the DSB (Fig. ), in some cases repair could be accomplished through strand invasion of a sister chromatid that has vastly greater homology on both sides of the DSB. A lack of Rad51 would leave only the SSA oligonucleotide mechanism of repair. To establish that oligonucleotide-mediated repair can substitute for a strand invasion mechanism, RAD+
-null diploids that had the I-SceI DSB-generating cassette in the TRP5
gene on chromosome VII (37
) were utilized. The TRP5
gene of the homologous chromosome was deleted by gene replacement with LEU2
. Of the four possible mechanisms of DSB repair, strand invasion with a sister or homolog, NHEJ, or oligonucleotide-mediated SSA, only the last would result in Trp+
cells (Fig. ). The SSA repair would simultaneously result in CORE loss, as would recombination with the homolog, or complete loss of the broken chromosome. Repair by NHEJ or by strand invasion with the sister chromatid would not result in loss of the CORE.
FIG. 2. Oligonucleotide versus homologous chromosome-mediated DSB repair in diploid cells. (A) The diagram shows the position of the CORE-I-SceI cassette and the DSB in TRP5 in one copy of chromosome VII. TRP5 has been replaced with the LEU2 gene in the second (more ...)
As previously reported (37
), the efficiency of DSB repair (frequency of Trp+
) by a mix of e
oligonucleotides in wild-type diploid cells was 30-fold less than that for recombinational repair with the homologous chromosome (CORE loss only) (Fig. ). The frequency of Trp+
transformants with e
, or both e
oligonucleotides was 9- to 40-fold less than that in a haploid wild-type parent (compare Fig. with Table S1A in the supplemental material for single oligonucleotides and with Table in reference 37
for the mixed oligonucleotides). However, the deletion of the RAD51
gene in the diploid strain resulted in a 15- to 30-fold increase in Trp+
colonies compared to that in the RAD+
strain. Interestingly, the frequency of Trp+
transformant colonies in the rad51
diploid cells was comparable to that found in the haploid rad51
mutant. There was no detectable loss of viability in the rad51
mutant cells (16% in rad51
versus 15% in wild-type cells, with or without oligonucleotides). Therefore, the increase in frequency of Trp+
colonies reflected the increase in efficiency of oligonucleotide-mediated DSB repair. The higher repair efficiency with the combined e
oligonucleotides than with the individual e
oligonucleotides in the wild type and rad51
mutant (for details, see reference 37
) is likely due to simultaneous annealing of complementary oligonucleotides to both sides of a DSB and/or to the formation of duplex oligonucleotide DNAs, which would be more resistant to degradation.
These results suggest that the presence of a homologous chromosome in the wild-type diploid cells provides an alternative pathway for the DSB repair, an option not available in the rad51 cells. Possibly, the opportunity to recombine with a homologous chromosome prevents additional rounds of cutting that might occur following repair between sister chromatids, thereby reducing the availability of a DSB for repair by the oligonucleotides. The increase in the frequency of oligonucleotide-targeted repair events in the absence of Rad51 could also be explained by Rad51 preventing oligonucleotide-directed annealing to ssDNA. We conclude that the Rad52 SSA oligonucleotide pathway for DSB repair is an efficient alternative to repair via strand invasion.
We note that SSA repair by an oligonucleotide(s) corresponded to at least 80% of the CORE loss events in the rad51
diploid (Fig. , see the table under the graph). The CORE loss colonies that are also Trp−
may be due to oligonucleotide-mediated repair with oligonucleotides that have mutations in the TRP5
) and/or to loss of the CORE markers independently from the oligonucleotides (e.g., deletion of the CORE region followed by telomere addition or chromosome fusion).
It is interesting that there is an overall reduction (as much as 80-fold) in the category of events in rad51 diploids that are due to only CORE loss. This is consistent with most CORE loss events in wild-type cells arising by strand invasion with the homologous chromosome. If in the absence of repair (strand invasion or SSA) the broken chromosome VII is lost, this either is a low-frequency event or leads to greatly reduced colony formation among cells with a DSB. (Note that a DSB occurs in ~30% of cells.)
Strand-biased oligonucleotide targeting to a site distant from a DSB.
As we previously demonstrated, a DSB can be repaired by oligonucleotides with homologies to sequences of up to 16 kb from the broken ends, suggesting that a DSB activates a large region for SSA recombination (37
). It is also known that a DSB can stimulate triparental recombination, promoting efficient gene conversion of a locus distant from the break in yeast diploid cells (32
). We hypothesize that in our system, such stimulation occurs via SSA. This is supported by the observation that in the absence of RAD51
, there was a 3.7-fold increase in repair frequency by single-strand oligonucleotides capable of generating a 16-kb deletion around the DSB (data not shown). These results in combination with the requirements for repair of the open versus the hairpin-closed DSB ends led us to propose that recombination distant from the DSB might be due to the appearance of ssDNA. This, in turn, predicts that ssDNA formed on either side of a DSB increases the likelihood of targeting oligonucleotides complementary to only one side of a break.
To test the idea of DSB-stimulated targeting to one side of a break, we initially constructed two diploid strains (Fig. ), where one chromosome VII contained a mutated TRP5 gene inactivated by a 31-base frameshift insertion (TRP5::ins31) and the other was the LEU2 replacement chromosome analogous to that presented in Fig. . The frequency of TRP5::ins31 reversions to Trp+ was <10−8. The CORE-I-SceI cassette was placed 10 kb upstream of the TRP5::ins31 target in one diploid strain (FRO-888) and 10 kb downstream of TRP5::ins31 in the other diploid strain (FRO-897). The induced DSB led to a 20- to 60-fold increase in targeting (Trp+) to the distant TRP5::ins31 site (Fig. ). We suggest that the homologous chromosome provided an opportunity for DSB repair in a manner similar to that for the experiments described in the legend for Fig. , and this enabled the events targeted to the side to be recovered. If targeting to the side of the break is stimulated by strand-specific resection (i.e., the 5′ to 3′ resection) there should be a strand bias since only the oligonucleotide that is complementary to the remaining strand would enable recombination. Indeed, the bias was observed in favor of the oligonucleotide complementary to the strand expected to remain intact after resection (Fig. ).
The findings for the diploid strains led us to examine the ability of a DSB to stimulate recombination at a distant site in yeast haploid cells under conditions where the DSB could be repaired directly. To further characterize resection and possible strand bias, we created a series of haploid strains (FRO-917) in which the CORE-I-SceI cassette, together with the I-SceI cutting site, was inserted ~6 (FRO-870), 10 (FRO-872), and 20 kb upstream (FRO-874) or 10 kb downstream (FRO-876) from the TRP5::ins31 locus (Fig. ). Oligonucleotide e or f, together with a second pair of oligonucleotides referred to as repairing oligonucleotides R1 and R2 (Fig. ) that would repair the DSB and delete the CORE-I-SceI cassette, was transformed into cells grown in glucose (i.e., no-break induction) or incubated for 7 h in galactose. As shown in Fig. , glucose-grown cells exhibited comparable low frequencies of Trp+ transformants for oligonucleotide e or f in all strains. In contrast, break induction led to a large increase in targeting with a strong strand bias in favor of the oligonucleotide that is complementary to the strand with a 3′ end if resection has occurred at the DSB. This was the f oligonucleotide when the break occurred upstream from the targeted TRP5::ins31 locus and was the e oligonucleotide for the downstream DSB. In the transformations where targeting to the side was stimulated by a DSB, the most Trp+ events were associated with CORE loss. The frequency of Trp+ events not associated with CORE loss was similar to the frequency of Trp+ events in the absence of a DSB (glucose). These findings are consistent with mechanisms that include the resection of a large region distant to the break during repair with oligonucleotides.
Interestingly, the presence of only one of the repairing oligonucleotides, R1 or R2, was sufficient to enhance strand-biased targeting. There was an approximate 7.5-fold increase in Trp+ transformants with the f oligonucleotide (Fig. ). However, there was no increased targeting by the e oligonucleotide.
The bias between oligonucleotides e
in targeted recombination distant from the DSB suggests 5′ resection (at least 20 kb) from the DSB, which is consistent with resection detected by physical means (13
). Importantly, in every transformant that arose from a break that was repaired by an oligonucleotide(s) and in which there was oligonucleotide correction of sequence up to 20 kb away, the restoration of the ssDNA to the double-strand state must have occurred. The sufficiency of just a single R1
oligonucleotide to repair the resected DSB can be explained only if two separate SSA events lead to the recovery of Trp+
recombinants. These findings support the view that a DSB can be repaired by a ssDNA via a “template” mechanism, as discussed below (Fig. ).
FIG. 4. Models of rejoining of DSB ends by ssDNA via two SSA events. Annealing interactions between cDNA regions are shown as dotted, red parallel lines; actual pairing is identified as red, parallel lines that are vertical; DNA synthesis is shown as a dotted (more ...)