Enhanced stimulation of chromosomal translocations and sister chromatid exchanges by either HO-induced double-strand breaks or ionizing radiation in Saccharomyces cerevisiae yku70 mutants

Abstract

DNA double-strand break (DSB) repair occurs by homologous recombination (HR) or non-homologous endjoining (NHEJ). In Saccharomyces cerevisiae, expression of both MATa and MATα inhibits NHEJ and facilitates DSB-initiated HR. We previously observed that DSB-initiated recombination between two his3 fragments, his3-Δ5′ and his3-Δ3′∷HOcs is enhanced in haploids and diploids expressing both MATa and MATα genes, regardless of the position or orientation of the his3 fragments. Herein, we measured frequencies of DNA damage-associated translocations and sister chromatid exchanges (SCEs) in yku70 haploid mutants, defective in NHEJ. Translocation and SCE frequencies were measured in strains containing the same his3 fragments after DSBs were made directly at trp1∷his3-Δ3′∷HOcs. Wild type and yku70 cells were also exposed to ionizing radiation and radiomimetic agents methyl methanesulfonate (MMS), phleomycin, and 4-nitroquinolone-1-oxide (4-NQO). Frequencies of X-ray-associated and DSB-initiated translocations were five-fold higher in yku70 mutants compared to wild type; however, frequencies of phleomycin-associated translocations were lower in the yku70 haploid mutant. Frequencies of DSB-initiated SCEs were 1.8-fold higher in the yku70 mutant, compared to wild type. Thus, DSB-initiated HR between repeated sequences on non-homologous chromosomes and sister chromatids occurs at higher frequencies in yku70 haploid mutants; however, higher frequencies of DNA damage-associated HR in yku70 mutants depend on the DNA damaging agent.

1. Introduction

Double-strand break (DSB) repair can occur by homologous recombination (HR) or non-homologous end joining (NHEJ). HR for DSB repair requires an undamaged chromatid, either a sister or a homolog, as a template for copying DNA, while NHEJ ligates ends of DSBs that have little or no homology. Genes that participate in both pathways show a remarkable degree of conservation from yeast to higher eukaryotes. Both pathways use Mre11, Rad50 and Xrs2 (NBS1), also called the MRX complex (for review, see [1]), which facilitates synapsis and processing of DSB ends to form single-stranded 3′ overhangs [2]. These 3′ single-stranded regions can serve as a substrate for proteins encoded by the RAD52 epistasis group genes, including RAD51, RAD55, RAD57, which catalyze synapsis and strand invasion of the undamaged sister chromatid or homolog with the broken end [3]. The resulting structure then serves to prime DNA synthesis across the gap generating Holliday intermediates that can be resolved to generate crossover and non-crossover events. MRX complex also participates in NHEJ by facilitating synapsis and subsequent ligation of both ends of the DSBs [1]. Proteins that participate in NHEJ include ku70 and ku80, Lig4 (ligase) [4], and in mammalian cells but not yeast, the catalytic subunit of DNA dependent protein kinase (DNA Pkcs) [5]. Because DSB ends are degraded, NHEJ is potentially mutagenic and can result in the loss of genetic material; whereas, HR repair of DSBs may result in the accurate repair of genetic material [5].

In mammalian cells, DSB repair occurs more frequently by NHEJ than HR, while HR is the preferred pathway for DSB repair in Saccharomyces cerevisiae (budding yeast). As a consequence, mammalian cell lines deficient in NHEJ, such as the chinese hamster ovary (CHO) V3 cell line [6], are hypersensitive to X-rays, while yeast yku70 mutants are only X-ray sensitive when RAD52 (HR) is eliminated [7]. In mammalian cell lines deficient in NHEJ, HR is enhanced after DSBs are generated by site directed endonucleases [8]. HR repair may be preferred in mammalian cells when DSBs occur as a consequence of replication fork collapse, as induced by aphidicolin treatment in S phase, in which there is only one end of the DSB [9]. DNA-dependent PKcs may suppress HR when DSBs are made in either G1 or S phase [10] by phosphorylation of Ataxia Telangiectasia Mutated (ATM) [11] and p53 [12].

In yeast, NHEJ pathway is repressed by expression of both MATa and MATα [13,14]), and thus, HR is enhanced between chromosome homologs in G₁ diploid cells. MAT heterozygosity represses the expression of NEJ1 [15], which encodes a protein required for NHEJ [13,16]. However, a/a diploids and α/α diploids are still more X-ray resistant than a or α haploids [17,18], respectively, implying that ploidy is also a factor in DSB-repair mediated by HR. MAT heterozygosity or diploidy does not enhance either spontaneous or X-ray-associated recombination between sister chromatids, which are preferred substrates for DNA repair [19,20], but does increase spontaneous and X-ray-associated recombination between repeats on non-homologous chromosomes [17]. Besides repressing NHEJ, a/α cells express higher levels of RAD51 and RAD54 [21]; and thus other factors under a/α control may insure that HR is the preferred DSB repair pathway. These observations suggest that ploidy, mating type, and phase of the cell cycle are all factors that shift the balance of DSB repair between HR and NHEJ pathways.

yku70 and yku80 mutants, defective in NHEJ, exhibit higher frequencies of particular recombination events that are initiated after HO digestion at cut sites placed at ectopic locations. For example, yku70 haploid and a/a diploid mutants exhibit modestly higher frequencies of DSB-initiated intrachromosomal gene conversion events and allelic recombination, respectively, compared to wild type [22]. These recombination events occur predominantly by gene conversion that is not associated with crossovers [22]. However, yku70 and yku80 mutants exhibit lower frequencies of spontaneous, MMS- and X-ray-associated deletions that are generated by recombination between repeats [23]. These deletions may occur by RAD51-independent mechanisms involved in single-strand annealing (SSA) [24]. Thus, enhanced HR in yku70 mutants may depend on the recombination assay and the DNA damaging agent used to stimulate the HR event.

Besides defective NHEJ, yku70 mutants also fail to resume cell cycle progression or adapt after cell cycle arrest is triggered by a single DSB [25]. DSBs signal G2 arrest when single-strand binding protein (RP-A) binds to single-stranded DNA, which occurs by the action of nucleases if NHEJ does not repair the DSB [25]. Since adaptation precedes recombination for some homology-directed rearrangements [26], it is unclear whether yku70 mutants may exhibit enhanced frequencies of DSB-associated chromosomal rearrangements.

We had previously observed that a/α diploid cells exhibit higher levels of X-ray-associated homology-directed translocations, compared to a/a and α/α diploids [17]. Recombination occurring between his3 fragments on sister chromatids or non-homologs that is initiated by HO endonuclease-generated DSBs is increased in haploids expressing both MATa and MATα [27]. It would thus be logical that MAT heterozygosity increases DSB-initiated recombination by suppressing NHEJ. However, several questions remain. First, it is unclear whether yku70 mutants would exhibit higher frequencies of X-ray-associated HR, since yKU70 does not confer X-ray resistance. Second, it is unclear whether the mechanism for increasing DSB-associated SCE in a/α haploids results from suppressing NHEJ, since NHEJ may already be suppressed in S phase.

In this manuscript, we asked whether yku70 mutants exhibit higher levels of DSB-initiated SCE and translocations. We observed five-fold higher frequency of DSB-associated translocations in haploid yku70 mutants, and a 1.8-fold higher DSB-associated SCE frequency in yku70 mutant, compared to wild type. For diploid strains, we found no enhanced frequency of translocations, consistent with the idea that NHEJ does not contribute to suppressing homologous recombination in yeast diploid cells.

2. Materials and methods

2.1. Strains and media

Standard media, including yeast extract, peptone, dextrose (YPD) and synthetic complete lacking histidine (SC-HIS) have been previously described [28]. Ura⁻ isolates (5-fluoro-orotic acid resistant [FOA^r]) were isolatated on FOA medium [28]. The strains used to measure homology-directed translocations and sister chromatid exchange (SCE) are listed in Table 1. These strains contain two truncated fragments of his3, his3-Δ3′ and his3-Δ5′ [19], as illustrated (Fig. 1). Directed translocations were measured in haploids and diploids, which were derived from one haploid containing the his3 fragments and one haploid lacking the truncated fragments.

Table 1.

Yeast strains

Lab name	Genotypea	Source
YA102	MATa ura3-52, his3-Δ200, ade2-101, 1ys2-801, trp1-Δ1, gal3−	M. Carlson
YB109	MATα ura3-52, his3-Δ200, ade2-101, 1ys2-801, trp1-Δ1, gal3− leu2-3, 112 GAL1∷his3-Δ5′, trp1∷his3-Δ3′∷HOcs, lys2− (leaky)	This laboratory
YB204	MATα ura3-52, his3-Δ200, 1ys2-801, trp1-Δ1, gal3− leu2-Δ1 trp1∷[his3-Δ3′∷HOcs, his3-Δ5′]	This laboratory
YB110	YB109 × YA102	This laboratory
YB356	MATα ura3-52, his3-Δ200, ade2-101, 1ys2-801, trp1-Δ1, gal3−	yku70∷URA3
	leu2-3,112,GAL1∷his3-Δ5′, trp1∷his3-Δ3′∷HOcs, lys2− (leaky),yku70∷URA3	Disruption in YB109
YB357	MATa yku70	FOAr isolate derived from yku70∷URA3 disruption in YA102
YB358	YB356 × YB357
YB163	MATa-inc trp1∷[his3-Δ3′∷HOcs, his3-Δ5′	This laboratory
YB359	MATa-inc trp1∷[his3-Δ3′∷HOcs, his3-Δ5′] yku70	This laboratory

^aGenotype the same as YA102 unless indicated.

Fig. 1.

Recombination assays used in this study. Ovals represent centromeres and lines represent chromosomes. For simplicity, the left arms of chromosomes are not included. The position and orientation of the his3 recombinational substrates are shown that are present in strains used to measure (A) reciprocal translocations; (B) unequal SCE. An “X” designates potential sites of crossovers, and the resulting chromosomal rearrangement is presented. An arrow and feathers denote HIS3. As indicated on the bottom of the figure, the 5′ deletion lacks the feathers and the 3′ deletion lacks the arrow. The two regions of sequence identity shared by the his3 fragments are indicated by decorated boxes; broadly spaced diagonal lines indicate a region of ~300 bp, and tightly spaced diagonal lines indicate a region of 167 bp.

To make yku70∷URA3 null mutants, we digested plasmidpHDFKO [29] with NotI, and selected for Ura⁺transformants [30] in strains YB109 and YA102. To confirm the phenotype of the yku70∷URA3 deletion, we determined whether the Ura⁺ transformants were temperature sensitive and whether they were defective in religating a linearized circular plasmid [31]. We confirmed the physical presence of yku70∷URA3 by detecting a ~2.1 kb PCR-generated fragment using primers 5′-GAACTTCTAATATATTCTGTG-3′ and 5′-ACCCTCTACCTTAGCATCCC-3′that anneal to YKU70 promoter and URA3, respectively. A Ura⁻ isolate (YB357) was obtained on FOA medium from the yku70 mutant derived from YA102. Ura⁺ Leu⁺ yku70 diploid mutants to measure DSB-initated translocations were selected after mating yku70 haploid mutants, YB356 and YB357. To measure DSB-initiated SCE in a MATa-inc yku70 strain, a MATα yku70 segregant of a diploid cross of YB357 and YB204 was first obtained. YB359 is a yku70 MATa-inc meiotic segregant derived from a diploid cross of MATαyku70 and YB163 and contains trp1∷[his3-Δ3′ ∷HOcs, his3-Δ5′].

2.2. DNA damaging agents

Compounds used in this study included methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), and phleomycin. Chemicals were purchased from Sigma or Aldrich Chemicals. Chemicals were dissolved in either dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) or water, depending on the specifications provided by the vendor.

2.3. Determining rates of spontaneous recombination and frequencies of DNA damage-associated recombination

The rates of spontaneous recombination were determined by the method of the median [32], as described by Esposito et al. [33]; 11 independent colonies were used for each rate calculation. We used the Mann–Whitney U-test [34] to determine the statistical significance of rate differences. The protocols used to measure DNA damage-associated recombination after exposure to MMS, 4-NQO, UV, and X-rays have been previously described [17,35]. The X-ray radiation source was purchased from Faxitron Inc. (Wheeling, IL), and the dose rate was 100 rad/min. The γ-ray source was a Nordion 1.8 kCi ¹³⁷Cs irradiator at 6.9 krad/h. A 254 nM germicidal lamp (2 J/m²/s) was used for UV irradiation. After irradiation, cells were washed with sterile H₂O and plated directly onto selective medium (SC-HIS) and the appropriate serial dilution was plated on YPD medium to measure viability.

We exposed cells to concentrations of chemicals that exhibit equivalent levels of lethality in both wild type and yku70 strains (Table 2). At least three independent experiments were done for each DNA damaging agent. The significance of the differences between yku70 mutants and wild type strains was determined using the two-tailed t-test [34].

Table 2:

HO endonuclease-stimulated translocation frequencies in wild type and yku70 mutants

Genotype (strain)a	%Viability after HO inductionb	His+ recombinants/Trp+ CFU × 105: before HO inductionc, after HO inductiond		Ratioe	%Trp+ CFU/total CFU (%): before HO induction, after HO induction
Haploid
Wild type (YB109)	27 ± 2.1	7.4 ± 8	48 ± 12	6.4	95 ± 0.7	83 ± 1.4
yku70 (YB356)	7.8 ± 2.7	5.0 ± 5.3	285 ± 156	57	95 ± 0.7	57 ± 17
Diploid
Wild type (YB110)	97 ± 3.8	1.4 ± 1	24 ± 1.4	17	95 ± 2.5	82 ± 8
ku70/ku70 (YB358)	90 ± 18	1.3 ± 1.4	29 ± 4.3	22	88 ± 5	78 ± 5

^aFor complete genotype, see Table 1.

^bTrp⁺ CFU after HO induction/Trp⁺ CFU before HO induction × 100%.

^cHis⁺ recombinants before HO induction/Trp⁺ CFU before HO induction.

^dHis⁺ recombinants after HO induction/Trp⁺ CFU after HO induction.

^eHis⁺ frequency after HO induction/His⁺ frequency before HO induction.

2.4. Induction of HO endonuclease

The galactose-inducible HO gene present in plasmid pGHOT-GAL3 [36] was introduced into the designated strains by selecting for Trp⁺ transformants. Trp⁺ isolates were then cultured at 30 °C in liquid SC-TRP and diluted in YPL. At a density of approximately 10⁷ cells/ml (mid log), glucose or galactose was added to a final concentration of 2%, to either repress or induce, respectively, the expression of HO endonuclease. After 2-h incubation at 23 °C, cells were directly plated onto SC-HIS to measure SCE formation and onto YPD to measure viability. Colonies were replica plated onto SC-TRP to measure the stability of pGHOT-GAL3.

2.5. Single-strand annealing (SSA)

To measure SSA frequencies, we measured the frequencies of his3 deletions resulting from HO-induced DSBs targeted at trp1∷his3Δ-5′his3Δ-3′∷HOcs. SSA creates one single his3 fragment, his3-Δ (5′, 3′) that lacks the HO cut site and both 3′ and 5′ ends of his3; isolates containing this deletion can no longer generate His⁺ recombinants. YPD isolates were patched onto YPD and replica plated onto SC-HIS and SC-TRP to count colonies that papillated to His⁺ and retained pGHOT-GAL3, respectively. In 10 Trp⁺ colonies that did not papillate to His⁺, we confirmed the presence of his3-Δ (5′, 3′) by detecting a 1.4 kb PCR product using primers 5′-CACGGCAGAGACCAATCAGTA-3′ and 5′-GCACTCCTGATTCCGCTAATA-3′ as previously described [37].

2.6. CHEF gels

Electrophoretic karyotypes of His⁺ recombinants were determined by pulse field electrophoresis. Undigested yeast DNA was resolved by contour-clamped homogeneous electric fields (CHEF), using 220 V (6 V/cm) for 26 h at 90 s. pulse time [36].

3. Results

The purpose of this study was to determine whether frequencies of DNA damage-associated translocations and SCEs were higher in yku70 mutants, compared to wild type. The recombination assays used in this study are shown in Fig. 1. The strains used to measure homology-directed translocations contain the his3-Δ5′ substrate on chromosome IV and the his3-Δ3′∷HOcs on chromosome II, while the strains used to measure unequal SCE contain the two his3 fragments in tandem at the trp1 locus, as previously described [36]. Upon induction of HO endonuclease, a DSB is made directly at his3-Δ3′∷HOcs. We measured rates of spontaneous translocations in both yku70 and wild type strains and found that the rate of spontaneous translocations was increased in the mutant to 6.7 × 10⁻⁸ from 2 × 10⁻⁸. We then compared the recombination frequencies in wild type and yku70 haploids and diploids after induction of HO endonuclease and after exposure to radiation and radiomimetic chemical agents.

3.1. Double-strand breaks (DSBs) generate more homology-directed translocations in haploid yku70 mutants than in wild type

To determine whether HO endonuclease-initiated DSBs stimulated more translocations in yku70 mutants, we introduced the pGHOT-GAL3 plasmid in both wild type (YB109) and yku70 haploid (YB356) strains by selecting for Trp⁺ transformants. We found that the percent of viable cells and pGHOT-GAL3 retention after HO endonuclease induction were threefold and approximately two-fold greater in wild type compared to yku70, indicating that HO-induced DSB at trp1∷his3-Δ3′∷HOcs conferred more lethality in the haploid yku70 mutant (Table 2). We observed a six-fold greater increase in translocation frequencies in the yku70 haploid mutant than in the wild type strain (Table 2). These results demonstrate that NHEJ is important in repairing the HO-induced DSB at trp1∷his3-Δ3′∷HOcs and that the HO endonuclease-induced DSBs can generate more homology-directed translocations in mutants defective in NHEJ.

We also determined whether HO endonuclease-initiated DSBs generate more translocations in ku70 diploid mutants. We introduced the pGHOT-GAL3 plasmid in wild type (YB110) and yku70 (YB358) diploid mutants. After HO endonuclease induction in wild type and yku70 diploids, we observed the same percent viability and translocation frequencies (Table 2). The frequencies of DSB-initiated translocations in the yku70 and wild type haploid mutants were higher than those found in the YKU70 a/α diploids, consistent with previous results [27]. These results demonstrate yKU70 does not suppress DSB-initiated translocations in diploid strains and that mating type heterozygosity likely increases frequencies of DSB-initiated translocations by repressing NHEJ.

3.2. X-ray-associated translocation frequencies are higher in haploid yku70 mutants

To determine whether radiation stimulates more translocations in yku70 compared to wild type, we exposed log phase haploid cells to 8 krad of X-rays and 13.8 and 20.7 krad of γ-rays. We observed that frequencies of X-ray-associated translocations were higher in yku70 haploid mutant, compared to wild type, while the yku70 mutant was as X-ray resistant as wild type (Fig. 2). These results are consistent with the idea that yKU70 does not contribute to X-ray resistance in strains competent at HR. We observed a five-fold to sixfold higher frequency of ionizing radiation-associated translocations in yku70 mutants compared to wild type (Fig. 2). We did not observe any difference in UV-associated recombination between wild type and yku70 (data not shown). These results indicate that X-rays stimulate more translocations in the yku70 mutants than in wild type strains.

Fig. 2.

Translocation frequencies (left) and percent survival (right) in yku70 (YB356, diagonal shading), and wild type (YB109, solid black shading) strains after exposure to X-rays (8 krad) and γ-rays (13.8 and 20.7 krad).

We then determined whether the X-ray radio-mimetic agents MMS and phleomycin and the UV-mimetic agent 4-NQO stimulate more translocations in yku70 compared to wild type (Table 3). Log phase cells were exposed to these DNA damaging agents and translocation frequencies and viability were determined. We observed no difference in viability after cells were exposed to these DNA damaging agents. We observed no difference in the frequencies of MMS-associated and 4-NQO-associated translocations in yku70 and wild type haploid strains; however, frequencies of phleomycin-associated translocations were higher in wild type than in yku70 mutants. These results demonstrate that the enhanced recombination in yku70 mutants depends on the DNA damaging agent.

Table 3.

Frequencies of translocations after exposure to MMS, 4-NQO and phleomycin in wild type and yku70 mutants

Genotype (strain)a	Frequency of His+ recombinants × 108b
	MMS (%viability)		Phleomycin (%viability)		4-NQO (%viability)
	0 ul	10 mM (0.1%)	0 ul	10 μM	0 μl	1 μM	10 μM
YKU70 (YB109)	5.3 ± 0.5	240 ± 30 (74)	5.3 ± 0.5	140 ± 64 (42)	<1	64 ± 12 (93)	720 ± 84 (52)
yku70 (YB356)	2.8 ± 1.2	270 ± 12 (74)	3.3 ± 1.3	41 ± 21 (60)	7.4 ± 6	49 ± 18 (92)	374 ± 84 (61)

^aFor complete genotype, see Table 1.

^bHis⁺ recombinants/CFu; n > 3.

3.3. Electrophoretic karyotypes of DNA damage-associated His⁺ recombinants

We previously observed that most spontaneous His⁺ recombinants from wild type contain reciprocal translocations, CEN2∷IV and CEN4∷II [19]. We therefore characterized the electrophoretic karyotypes of spontaneous and X-ray-associated translocations from wild type and yku70 haploids (Fig. 3). We observed that four out of four spontaneous His⁺ recombinants from wild type and three out of four spontaneous His⁺ recombinants from yku70 haploid strains contain reciprocal translocation. After exposure to 13.8 and 20.7 krad of ionizing radiation, we characterized the electrophoretic karyotype of seven His⁺ recombinants from wild type and eight from the yku70 strain. All eight His⁺ recombinants from the yku70 strain contained reciprocal translocations. We observed that three X-ray-associated His⁺ recombinants from wild type contained reciprocal translocations, while one contained non-reciprocal translocations, and three others contained atypical rearrangements. This data suggest that X-ray-associated rearrangements in yku70 mutants predominately occur by homologous recombination, whereas NHEJ may participate in the formation of X-ray-associated rearrangements in wild type.

Fig. 3.

Electrophoretic karyotype of γ-ray-associated His⁺ recombinants generated in the haploid wild type (YB109). (A) YB109 His⁻parent; (B) His⁺ recombinants containing CEN2∷IV and other translocation; (C) His⁺ recombinant containing other rearrangements; (D) His⁺ recombinant containing reciprocal translocation; (E) YB109 His⁻ parent. Arrows indicate the positions of chromosomes II and IV, and translocations CEN2∷IV and CEN4∷II.

3.4. Double-strand breaks (DSBs) generate more SCEs in haploid yku70 mutants than in wild type

We previously observed that expression of MATa and MATα enhances DSB-initiated SCE events [27]. These results imply that repressing NHEJ will increase homologous recombination not only in the G1 stage but also in the G2 stage of the cell cycle. We introduced pGHOT-GAL3 in wild type (YB163) and in the yku70 (YB359) mutant, and measured SCE frequencies and viability after the induction of HO endonuclease (Table 4). We did not observe any significant decrease in viability after HO endonuclease was induced (P = 0.1). However, SCE frequencies were 1.8-fold higher in yku70 mutant, compared to wild type (P < 0.05). These results indicate that yku70 mutations confer a higher frequency of DSB-initiated SCE.

Table 4.

HO endonuclease-stimulated SCE frequencies in wild type and yku70 mutants

Genotype (strain)a	%Viability after HO inductionb	His+ recombinants/Trp+ CFU × 105: before HO inductionc, after HO inductiond		Ratioe	Trp+ CFU/total CFU (%): before HO induction, after HO induction
Haploid
Wild type (YB163)	78 ± 15	6.5 ± 3	79 ± 16	12	95 ± 0.7	83 ± 1.4
yku70 (YB359)	67 ± 11	12 ± 7	143 ± 45	12	88 ± 5	91 ± 2

^aFor complete genotype, see Table 1.

^bTrp⁺ CFU after HO induction/Trp⁺ CFU before HO induction × 100%.

^cHis⁺ recombinants before HO induction/Trp⁺ CFU before HO induction.

^dHis⁺ recombinants after HO induction/Trp⁺ CFU after HO induction.

^eHis⁺ frequency after HO induction/His⁺ frequency before HO induction.

3.5. Frequencies of intrachromosomal deletions are the same in yku70 and wild type

Because DSBs at trp1∷his3-Δ5′ his3-Δ3′∷HOcs can initiate both sister chromatid recombination and SSA [37], we also measured DSB-initiated SSA in wild type (YB163) and yku70 (YB359) strains. SSA annealing generates a deletion between the two his3 fragments that results in one truncated his3 fragment (Fig. 4). Strains containing the deletion can no longer generate His⁺ recombinants due to SCE. We therefore measured the percentage of cells that can no longer generate His⁺ recombinants in wild type and in yku70 mutants. We observed that 72 ± 4% (n = 3) could generate deletions in yku70 mutants, a percentage that was similar to wild type (75% [37]). These results indicate that yku70 mutants are not defective in SSA between two closely linked repeated sequences. Since SSA is the predominant mechanism for DSB repair at trp1∷his3-Δ3′ his3-Δ5′, these results are consistent with observations that induction of HO endonuclease does not confer higher levels of lethality in yku70 mutants, compared to wild type.

Fig. 4.

SCE initiated by a HO-induced DSB can initiate SSA, SCE and NHEJ in wild type and yku70 mutants. DSB repair can occur either by SSA or unequal SCE. Symbols representing his3-Δ5′ and his3-Δ3′ are described in the legend to Fig. 1. “A” and “B” are two sister chromatids. The left panel shows repair of the HO endonuclease-generated DSB by the SSA pathway, which generates intrachromosomal deletions and a his3 fragment lacking both the 5′ and 3′ ends. Right panel of the figure shows repair of the HO endonuclease-induced DSB by homologous recombination. Unequal SCE generates HIS3 as shown at the bottom right. SCE events are measured by selecting for His⁺ recombinants while intrachromosomal deletions are screened, since the deletion renders cells unable to generate His⁺ recombinants.

4. Discussion

In budding yeast, the choice between HR and NHEJ mechanisms for DSB repair can be influenced by ploidy, phase of the cell cycle, and MAT heterozygosity. We had previously observed that MAT heterozygosity enhances radiation-associated recombination between his3 fragments on non-homologous chromosomes [17] and recombination between his3 fragments initiated by HO endonuclease-generated DSBs, regardless of the orientation or location of the fragments [27]. Here, we observed that yku70 haploid mutants, defective in NHEJ, exhibit five-fold higher frequencies of DSB-associated translocations and 1.8-fold increase in DSB-initiated SCEs; yku70 diploid mutants did not exhibit a higher frequency of DSB-initiated translocations. Compared to wild type, we observed higher frequencies of X-ray-associated translocations, but not 4-NQO-, MMS-and phleomycin-associated translocations in yku70 haploid mutants. These results support the following conclusions: First, NHEJ represses the DSB-initiation of homology-directed chromosomal rearrangements in haploid cells. Second, yku70 mutations can confer higher frequencies of DSB-initiated HR in G₂ cells. Third, enhanced frequencies of DNA damage-associated translocations in yku70 mutants depend on the type of recombinogenic lesion. Our results extend previous observations that frequencies of both allelic recombination and intrachromosomal gene conversion events are enhanced in yku70 mutants [22] and indicate that yKU70 can also reduce genetic instability by decreasing frequencies of chromosomal rearrangements that result from reciprocal exchange.

Our results are consistent with ideas that expression of MATa and MATα enhances DSB-initiated recombination between his3 fragments by repressing NHEJ [14]. Frequencies of DSB-initiated translocations in the yku70 haploid were significantly higher than in either wild type or yku70 diploids, possibly because of the high level of lethality that is conferred by HO-induced DSBs in the absence of a chromosomal homolog. Compared to the MATa/MATα diploid, MATa/MATα yku70 diploid mutants did not exhibit higher levels of DSB-initiated translocations since NHEJ was already repressed. Considering that MAT heterozygosity confers higher expression of RAD51 and RAD54 [21], it is also possible that there are additional mechanisms by which MAT heterozygosity or ploidy can enhance recombination.

We speculate that there are two reasons why yku70 mutants exhibited higher frequencies of DSB-initiated SCE and translocations but not higher frequencies of DSB-initiated intrachromatid deletions that likely occur by SSA. First, SSA is highly efficient in repairing DSBs, while frequencies of DSB-initiated unequal SCE and translocations are three magnitudes lower than DSB-initiated SSA. Thus, an increase in recombination frequencies in yku70 mutants may be easier to detect when recombination frequencies in wild type are low. Second, the genetic requirements for SSA, translocations, and SCE are different [37]. Cervelli and Galli [23] have suggested that Ku70 and Ku80 may recruit recombination proteins required for both spontaneous and DSB-initiated SSA; such proteins may not be required for SCE and translocations.

Our studies indicate that NHEJ is the major pathway for the repair of the HO-induced DSB at trp1∷his3-Δ3′∷HOcs in the haploid strains containing GAL1∷his3-Δ5′. We previously observed that HO endonuclease-induced DSBs at trp1∷his3-Δ3′∷HOcs initiate recombination with GAL1∷his3-Δ5′ at low frequencies, and the majority of cells that contain the HO-induced DSB are inviable [27]. Here we observed a three-fold higher level of lethality after HO induction in yku70 haploid mutants. Clikeman et al. [22] observed lower DSB-induced lethality in yku70 mutants when HR was initiated between homologs, suggesting that higher levels of HR decrease the lethality of DSBs in yku70 mutants. We suggest that one reason why we did not observe lower DSB-induced lethality in ku70 haploid mutant is that the recombination frequency between his3 fragments is low, thus rendering it more difficult for the relative increase in recombination to affect viability. Nonetheless, we observed a five-fold increase infrequencies of DSB-initiated translocations in yku70, compared to wild type, indicating that the DSBs generated in yku70 are more recombinogenic than in wild type. We speculate that DSB-initiation of HR could result from the greater availability of single-stranded DNA in yku70 mutants for binding of recombination proteins, as suggested by Frank-Vaillant and Marcand [38].

The modest increase in DSB-initiated SCE events in yku70 mutants is consistent with other studies that NHEJ participates in DSB repair in both S phase and G2 phases of the yeast cell cycle. For example, yng2 mutations, which confer MMS sensitivity and deficiency in the S phase DNA damage response [39], are synthetically lethal with yku70 mutations. In addition, NHEJ precedes HR repair of HO-induced DSBs when DSBs are made in either G1 or G2, although resection of the DSB proceeds faster in S phase [38]. Thus, results obtained in S. cerevisiae are consistent with those obtained in mammalian cell culture that have suggested that both HR and NHEJ participate in DSB repair in G2 [8,9]. Our results, however, are the first to indicate that DSBs are more efficient initiators of SCE when NHEJ is deficient.

We observed that DNA damage-associated translocations were enhanced in yku70 mutants after exposure to X-rays but not after exposure to 4-NQO, phleomycin and MMS. We do not know the exact difference between the types of DNA damage to indicate why one DNA damaging agent should stimulate more translocations in yku70 mutants than another. We suggest that the differences between the various DNA damaging agents may depend on how DSBs are generated by these DNA damaging agents and whether they can be repaired by NHEJ. For example, HO endonuclease-induced DSBs can be repaired by NHEJ when made at sites other than the MAT locus [22,36], and we suggest that a subset of X-ray-induced DSBs can also be repaired by NHEJ, since X-ray resistance is lower in yku70 rad52 double mutants, compared to rad52 mutants [7]. However, DSBs may not be repaired by NHEJ if the DSB is formed by replication fork collapse in which only end of a DSB is present [8]. Since both MMS, 4-NQO and phleomycin trigger S phase checkpoints, we speculate that one reason that MMS and phleomycin do not stimulate more translocations in yku70 mutants is that DSBs induced by these agents may initiate recombination after replication fork collapse [40].

In summary, we observed that yku70 null mutation confers higher levels of DSB-initiated recombination events between repeats on non-homologous chromosomes and between sister chromatid, and that a subset of DNA damaging agents can stimulate more recombination in yku70 mutants, compared to wild type. Thus, yku70 mutations could potentially stimulate other reciprocal exchange events initiated between repeated sequences in yeast. It would thus be interesting to determine whether mutations in the mammalian orthologues of yKU70 and yKU80 could also increase radiation-associated HR events that generate chromosomal rearrangements.