gene switching in budding yeast can be induced synchronously in a population of cells by induction of a galactose-regulated HO endonuclease gene. For strain tGI268, when an HO-induced DSB is created in MATa
, the Ya
sequence is replaced by the 3.6-kb Yα-λ sequence copied from HMR
α-λ. Thus, this gene conversion event requires the synthesis of >3.6 kb of new DNA. The insertion of these additional sequences was needed in order to generate appropriately sized restriction fragments whose density would be well resolved by equilibrium density gradient centrifugation. The insertion of phage λ DNA did not affect the silencing of the HMR
locus, as HO endonuclease did not cut its cleavage site, which is occluded by positioned nucleosomes in this locus (28
; data not shown). To assess the nature of repair DNA synthesis, it was necessary to carry out the isotope incorporation under conditions in which normal DNA replication was not occurring. We studied HO-induced recombination in G2
-arrested cells, after replication was complete, rather than in G1
, prior to DNA replication, because Cdk1 kinase, which is required for completing HO-induced recombination, is inactive in G1
(Fig. ) (15
). Cells were grown in medium containing heavy isotopes of carbon and nitrogen (13
C and 15
N); after growth was arrested in G2
/M with nocodazole, the cells were shifted to medium containing normal, light isotopes. Thirty minutes after the shift in the medium, the HO endonuclease gene was induced by adding galactose to a final concentration of 2%, to induce a DSB at MATa
. Most of the cells (>90%) remained blocked in G2
during the 6-h duration of the experiment; therefore, incorporation of new nucleotides synthesized from light isotopes was limited to DNA synthesis during the gene conversion event. Southern blot analysis showed that the replacement of Ya
by Yα-λ DNA sequences at MAT
was completed within 6 h (Fig. ). Repair efficiency is only about 50% of that observed for HO-induced cycling cells.
By using CsCl equilibrium density centrifugation, we determined whether newly incorporated light-isotope-labeled (LL) DNA was incorporated at MAT
and/or at HMR
α::λ. The results of Southern blot analyses of fractions taken from the CsCl gradient are shown in Fig. . The position of LL DNA was determined from MAT
α::λ cells grown in normal 12
N medium, and the position of semiconservatively replicated DNA from the same cells but grown first in heavy (13
N) medium and then allowed to go through one round of DNA synthesis in light (12
N) medium (HL DNA) was determined. It appears that all the newly synthesized DNA is located in the recipient, because the MAT
α::λ cell DNA sediments at nearly the same position as LL DNA isolated from cells grown in normal medium (Fig. ). The small shift of the newly created MAT
α-λ fragment toward the HL peak probably results from the fact that the 3.7-kb BamHI-StyI fragment that we tested is 0.7 kb longer than the newly synthesized 3.0-kb Yα-λ sequence (Fig. ). The MAT
Z region and more-distal sequences are partially resected and later will be filled in by light (12
N) nucleotides, but the strand ending 3′ at the HO cut site is not replaced (37
); thus, the BamHI-StyI fragment would be expected to be about 17% heavier than a fully LL segment.
In contrast, after completion of MAT switching, the HMRα-λ fragment remained fully heavy isotope labeled (HH) and sedimented in the same fractions as it did before repair, implying that no newly synthesized DNA was inherited by the donor locus (Fig. ). The donor fragment analyzed is 7.6 kb long, and only about 3.3 kb participates in recombination (Yα-λ plus Z), so that only about half of the region was likely to have been replicated during repair (Fig. ). Therefore, about 50% of one strand of the donor molecule would be light if MAT switching occurred by the dHJ mechanism. Consequently, if the dHJ mechanism were used, about 25% of the total DNA would be LL, and we should observe a peak halfway between HL and HH for the donor. Because we do not observe this shift, we conclude that the molecular weight of the donor did not change during recombination. We note, however, that nocodazole arrest of growth is not perfect, and a certain percentage of cells escape the arrest; consequently 5% of HMRa-λ fragments sedimented as HL (lighter than we would expect from the dHJ model of recombination). To make sure that this was due to the escape from nocodazole arrest and is not relevant to the repair, we tested the sedimentation of DNA fragments with two other loci on the same chromosome (THR4 and HIS4) by hybridizing the same membrane (sampled 6 h after DSB induction) with THR4- and HIS4-specific probes. In both cases, we observed a very small fraction of HL DNA due to escape from nocodazole arrest, similar to the results with the HMRα-λ fragment (Fig. ).
Our results show clearly that both DNA strands that are newly synthesized during DSB repair are recovered in the recipient locus. This finding is not compatible with a dHJ mechanism involving an HJ resolvase in which almost all of the resolved recombinants are recovered as noncrossovers. Such a mechanism would produce HL restriction fragments for both the donor and the recipient, contrary to what was observed. These data provide strong evidence that either SDSA or a dHJ unwinding model is the predominant repair process in mitotic cells. Based on results from previous studies, we suggest that both pathways are used but that the predominant one is SDSA. In particular, SDSA best explains both the frequent expansion/contraction of repeated sequences copied into the recipient during gene conversion (27
) and the tripartite recombination events, where sequences homologous to each double-strand-break end are located on different chromosomes.
We note that, in order to use density transfer methods, we were obliged to study DSB repair that includes the copying of a larger nonhomologous donor sequence than the usual 700-bp segment, so that the new DNA was incorporated into a 3-kb fragment. Moreover, MAT
switching is inherently asymmetric, in that one side of the DSB has nonhomologous sequences that prevent the immediate engagement of an end that can prime new DNA synthesis. It is possible that when both ends are equally capable of strand invasion and initiation of new DNA synthesis and when there is no large heterologous region to be copied, a higher proportion of cells will use the dHJ mechanism. To address whether the exceptional features of MAT
switching create a special case in which SDSA predominates, we tested the ratios of crossovers and noncrossovers in three different ectopic recombination assays between chromosomes III and V, in which we altered the donor and recipient (Fig. ). The first assay showed that both DSB ends are almost perfectly homologous (there is a single-base-pair difference that prevents the MAT
donor from being cleaved by HO). The equivalence of the two ends might promote dHJ formation and an increased level of crossovers (Fig. ). The second assay showed that the recipient has one nonhomologous end (the 0.65-kb Ya
sequence) and the donor has a heterologous segment (the 0.7-kb Yα sequence) that needs to be copied to the recipient; this scenario perfectly imitates MAT
switching, except that the donor is on a different chromosome (Fig. ). The third assay showed that the recipient has two nonhomologous tails, 75 and 46 bp long, and a 708-bp hisG
insertion that needs to be copied during repair (Fig. ). If nonhomologous segments at the ends or a large heterologous segment at the donor locus would have any impact on the engagement of the two DSB ends and the formation of a dHJ crossover intermediate, then we would expect to see significant differences in crossover frequency in these three cases. The results of Southern blot analyses following DSB repair after HO induction are shown in Fig. . There is a delay in recombination when the two ends are nonhomologous, as we have seen before (4
). However, in all cases the exchange frequencies were the same, indicating that the selection of a noncrossover SDSA pathway or a crossover-generating dHJ pathway is not affected by nonhomologous tails or heterology in the donor. These new results are in agreement with our previous studies of HO-induced recombination when both ends are homologous to those of the donor (14
). Thus, it seems that MAT
switching is a representative function for studying DNA synthesis during DSB repair. Nevertheless, we acknowledge that it is still possible that recombination between fully homologous sequences, such as between two sister chromatids, can occur more often by the dHJ mechanism than by SDSA.
FIG. 4. Terminal nonhomology at the DSB ends and the presence of an insertion in the donor sequence have no impact on crossover and noncrossover pathway choice. Three different recombination scenarios were studied to check if terminal nonhomology (NH) or heterology (more ...)
The low level of crossing-over in mitotic cells suggests that dHJs are formed only infrequently, and probably about half of them are removed by Sgs1-Top3 activity (14
). In interchromosomal ectopic HO-induced recombination, only about 4% of cells repair the DSB with an accompanying crossover (14
). In MAT
switching, the proportion of crossovers that generate deletions between MAT
is also small (9
). Such a small proportion would not have been detected in these experiments. Crossing-over might reflect the operation of the dHJ mechanism, or it might indicate that sometimes SDSA mechanisms can “trap” HJs, as has been suggested for some variations of the SDSA mechanism (2
). Two enzymes that promote the noncrossover SDSA pathway are the Srs2 and Mph1 DNA helicases (14
; our unpublished data). We are currently investigating the roles of these two helicases in promoting noncrossover recombination pathways.
Previously, Arcangioli (3
) used density transfer to study mat
gene switching in Schizosaccharomyces pombe
. Approximately 25% of fission yeast cells switch mating types in any generation, and correlating with this proportion, about 25% of the newly synthesized mat
DNA was LL, suggesting that an SDSA mechanism was involved. This gene conversion event is fundamentally different from that examined here, in that there is no HO-like nuclease to make the DSB; rather, a DSB is generated during the S phase itself from a preexisting nick, and the repair process is dependent on cells being in S phase (11
). In the case we studied here, repair is initiated outside S phase and both strands of the Y region of MAT
must be copied de novo.