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Genetics. Jan 2005; 169(1): 51–63.
PMCID: PMC1448898

X-Ray Survival Characteristics and Genetic Analysis for Nine Saccharomyces Deletion Mutants That Show Altered Radiation Sensitivity


The availability of a genome-wide set of Saccharomyces deletion mutants provides a chance to identify all the yeast genes involved in DNA repair. Using X rays, we are screening these mutants to identify additional genes that cause increased sensitivity to the lethal effects of ionizing radiation. For each mutant identified as sensitive, we are confirming that the sensitivity phenotype cosegregates with the deletion allele and are obtaining multipoint survival-vs.-dose assays in at least one homozygous diploid and two haploid strains. We present data for deletion mutants involving the genes DOT1, MDM20, NAT3, SPT7, SPT20, GCN5, HFI1, DCC1, and VID21/EAF1 and discuss their potential roles in repair. Eight of these genes cause a clear radiation-sensitive phenotype when deleted, but the ninth, GCN5, results in at most a borderline phenotype. None of the deletions confer substantial sensitivity to ultraviolet radiation, although one or two may confer marginal sensitivity. The DOT1 gene is of interest because its only known function is to methylate one lysine residue in the core of the histone H3 protein. We find that histone H3 mutants (supplied by K. Struhl) in which this residue is replaced by other amino acids are also X-ray sensitive, which confirms that methylation of the lysine-79 residue is required for effective repair of radiation damage.

YEAST mutants initially isolated on the basis of sensitivity to ionizing radiation (IR) have proved invaluable for understanding many aspects of DNA transactions in eukaryotes. Despite this, pathways and mechanisms of cellular recovery from IR damage are less well understood than many other aspects of DNA repair. To fully understand IR repair, it is important to identify all the genes involved. The availability of a genome-wide set of deletion mutants (Winzeler et al. 1999) now makes it possible to identify virtually all the nonessential genes in Saccharomyces that play a role in conferring resistance to ionizing radiation. We and others have begun a systematic search for such genes that were missed in classical screens of mutagenized cells. These screens, summarized by Game and Mortimer (1974), were apparently very effective in identifying mutants that confer a high degree of sensitivity. Few or no new genes have been found in the last 30 years whose mutants confer the extreme IR sensitivity of deletion alleles in the RAD51 epistasis group. However, classical screens were less effective at identifying mutants with moderate IR sensitivity, and a significant number of such mutants remain to be characterized (Bennett et al. 2001; Game et al. 2003). Mutants that combine a growth defect with modest radiation sensitivity can be especially hard to identify in replica-plating screens, since they will form small colonies whose sensitivity is hard to identify compared to poor growth on the control plate. In addition, assigning novel mutants in unknown genes to specific loci can be laborious. However, identifying a modest IR-sensitive phenotype regardless of growth rate is not difficult when pure cultures of known mutants are tested for X-ray sensitivity individually. Even modestly sensitive mutants are important in understanding repair, because they can uncover unrecognized or redundant pathways and mechanisms. Double- or multiple-mutant combinations may serve to expose the full role of such pathways.

We have been using the genome-wide deletion set (Winzeler et al. 1999) to identify new genes or open reading frames (ORFs) whose deletion alleles confer increased sensitivity to killing by X rays. Some preliminary information is already available about the IR sensitivity of many of the cataloged yeast deletion mutants. Bennett et al. (2001) screened 3670 diploid deletion strains in plate assays and identified 107 as potentially IR sensitive. However, follow-up tests were not done and the list includes some false positives where factors other than the deletion allele contributed to IR sensitivity (Game et al. 2003). In addition, >1000 new deletion strains have become available since the work of Bennett et al. (2001). Further information is available from a screen developed in the laboratories of J. Martin Brown and R. W. Davis at Stanford (see Birrell et al. 2001; Game et al. 2003). The screen utilizes a pool of nearly all the deletion mutants combined into one culture. It involves ranking the relative change in abundance, as judged by ratios of hybridization signals of unique bar code sequences to a microarray plate, of each mutant after irradiation and brief growth of the pool, compared to the starting abundance for that mutant. Mutants with enhanced sensitivity show less relative abundance in the pool after irradiation. Using this methodology >4600 mutants were ranked in order of relative abundance in a pool of mutants 18 hr after IR treatment compared to unirradiated controls (Game et al. 2003). However, ~9% of deletion mutants in this ranking were not adequately monitored for IR sensitivity using this method because they were insufficiently abundant in the pool prior to irradiation to allow adequate assessment of radiation effects (see Game et al. 2003).

We have been directly assaying IR sensitivity in some of the 1000 or so mutants that were not in the collection surveyed by Bennett et al. (2001), focusing especially on those that also could not be adequately ranked by the pool method (Birrell et al. 2001; Game et al. 2003). In addition, we have used quantitative survival assays in combination with genetic analysis to more rigorously test and characterize X-ray sensitivity in several other mutants that were identified as potentially sensitive either in the pool assay or by Bennett et al. (2001) or were identified by us as interesting on the basis of relationships to other known mutants. Secondary factors unrelated to the created deletions can sometimes be responsible for IR sensitivity in mutant strains from the genomic library (Game et al. 2003). These can include independent mutations, polyploidy, and in diploids, homozygosity for the mating type locus. Hence it is necessary to verify that the phenotype of each newly identified IR-sensitive mutant is truly conferred by the deletion. In addition, it is important to obtain survival curves for mutants initially identified on the basis of qualitative tests.

In this report, we present survival characteristics for nine deletion mutants, eight of which we have confirmed genetically to confer X-ray sensitivity. The ninth, gcn5Δ, may confer marginal sensitivity. Six of these mutants, involving deletions of the genes MDM20, DOT1, SPT7, SPT20, HFI1, and GCN5, were initially identified as sensitive in spot tests in our own laboratory. We chose the remaining three, deleted for the NAT3, VID21, and DCC1 genes, on the basis of previously reported sensitivity in spot tests (Bennett et al. 2001). We discuss the possible significance of these genes in DNA repair and also report difficulties in confirming the radiation sensitivity of two additional mutants. We also tested the mutants for cross-sensitivity to ultraviolet radiation, and we present data suggesting either no sensitivity or at most minor UV sensitivity.


Yeast strains:

We obtained a library of ~4700 individual haploid deletion strains in the α-mating type (background strain BY4742) from Research Genetics (Huntsville, AL; now Invitrogen Life Technologies). These deletion strains can also be obtained from EUROSCARF (Frankfurt, Germany). Genotypes of the parental yeast strain BY4742 and construction of the deletion strains have been described (Brachmann et al. 1998; Winzeler et al. 1999). Information is also available at the Saccharomyces Genome Deletion Project website, http://www-sequence.stanford.edu/group/yeast_deletion_project/deletions3.html. The prototrophic wild-type strains X2180-1A and X2180-1B were available to us from R. K. Mortimer. They are haploid spore clones derived from diploid X2180, which was identified by Mortimer as a spontaneous diploid MATa/MATα derivative of haploid strain S288C (see Mortimer and Johnston 1986). They are thus isogenic in background to the strains used to generate the deletions, which were derived from S288C by a series of transformations and subsequent background-isogenic crosses (Brachmann et al. 1998).

Genetic methods and media:

Genetic methods including tetrad dissection were as described by Sherman et al. (1982). Cultures were incubated at 30° unless a temperature-conditional phenotype was segregating, in which case 25° was used. Rich media (YPD) and supplemented minimal media were prepared as described by Sherman et al. (1982). To make inositol-less medium, we used yeast nitrogen base (YNB) without inositol, obtained from Q-biogene, in place of regular YNB. To induce meiosis, we incubated cultures for 4 or more days, usually at 30°, on solid Fogel's sporulation medium. This contains the following: 9.65 g potassium acetate, 1 g glucose, 2.5 g yeast extract (Difco, Detroit), and 2% agar made up to 1 liter and autoclaved. To score the geneticin-resistance phenotype, we used YPD plates supplemented with geneticin (obtained from Sigma, St. Louis) added from filter-sterilized solution shortly before pouring plates, to give a final concentration of 150 μg/ml.

Spot-testing the mutants for X-ray sensitivity:

We retrieved mutants from cold storage and grew cultures. We arrayed fresh cultures in thin patches of 20 per plate on YPD medium and replica stamped each plate immediately to six more YPD plates. We irradiated two plates from each set with 78 krad (7.8 Gy) and two more with 156 krad (15.6 Gy) of X rays. We incubated one plate from each pair at 25° and one at 37°, together with the unirradiated plates. We monitored each set at 1 and 2 days after irradiation for evidence of sensitivity in any mutant, as evidenced by less than average growth or survival compared to the unirradiated plate at the same temperature.

X-ray treatments:

For all X-ray exposures, we used a Machlett OEG 60 X-ray tube with a beryllium window and a Spellman power supply operated at 30 kV and 15 mA to deliver a dose rate of 130 rad/sec of “soft” X rays. For survival curves, unless otherwise noted, log-phase cells from overnight liquid YPD cultures grown at 30° were diluted appropriately in fresh YPD and grown for several more hours with vigorous shaking. When a cell density of 1 × 107 to 2.5 × 107 cells/ml was reached, as determined with a hemocytometer, serial dilutions were made in distilled water cooled in ice. Cells plated from the appropriate dilution were irradiated at room temperature on YPD plates, to yield (ideally) ~200 surviving colonies/plate, with two plates per dose. Cultures were inspected for clumpiness. If clumps were observed, cultures were sonicated briefly to disperse them. Colonies were counted after incubation for 5–6 days at 30° unless otherwise noted.

UV-radiation treatments:

Cells were prepared in log phase for UV survival curves as outlined above for X rays. They were irradiated on YPD plates using a shielded apparatus containing five General Electric G8T5 tubes giving most of their radiation at 254 nm. Plates were incubated in the dark and colonies were counted as for X-ray curves.


Spot-testing individual mutants:

To identify additional yeast genes that may be involved in IR repair, we began by assaying X-ray sensitivity among some of the 1000 or so mutants that were not in the collection surveyed by Bennett et al. (2001), focusing on those that also scored low or could not be adequately ranked by the pool method (Birrell et al. 2001; Game et al. 2003). We spot-tested 357 mutants selected by these criteria for sensitivity to X rays at 37° and at 25°, using two doses (78 krad/7.8 Gy and 156 krad/15.6 Gy) at each temperature. We also noted temperature effects on growth in the absence of radiation. It is important to assess IR sensitivity at more than one temperature, even in deletion mutants, since some yeast proteins play a role in repair that is temperature dependent (Lovett and Mortimer 1987).

Of the 357 mutant cultures initially tested, 25 were chosen for retesting from those that appeared to be the most convincing candidates for X-ray sensitivity at one or both temperatures in the first test. After these 25 were retested in similar plate tests, 3 mutants, the dot1, mdm20, and spt20 deletion strains, were chosen for further study. Three more mutants, those carrying the spt7, hfi1, and gcn5 deletions, were also chosen for study, on the basis of their known relationship (Grant et al. 1997; Horiuchi et al. 1997) to the identified spt20 mutant. Two of these, the spt7 and hfi1 deletion strains, showed sensitivity in spot tests. The third, the gcn5 deletion, showed no clear sensitivity in spot tests, but showed a possible mild sensitivity (less than the other mutants) in survival assays. These 3 mutants were also among the ~1000 deletions absent from the initial set. One was also absent from the pool of mutants studied in the Brown laboratory. The other 2 showed a borderline low score or a borderline low signal in the pool assay but were not selected in our initial list of 357 cultures.

In addition to the above six mutants, we chose to study five mutants reported by Bennett et al. (2001) to be sensitive to cesium-137 gamma rays. These were the deletions of NAT3, VID21 (ORF YDR359C), DCC1, HTL1, and DEF1 (VID31). We chose the five for which the authors presented the most convincing observations of sensitivity in a diploid strain and in both haploid parents. We confirmed that the deletion library strains listed as the MATα haploids that contained each of these five deletions were X-ray sensitive in spot tests.

Genetic analysis of mutants:

As shown previously (Game et al. 2003), some mutant strains from the genomic library contain secondary genetic changes in addition to the created deletion. To determine if the deletion by itself is both necessary and sufficient to confer X-ray sensitivity in the strains we studied here, we crossed each deletion mutant with a wild-type MATa strain. We determined whether the X-ray-sensitive phenotype cosegregated with the deletion in meiotic tetrads from these crosses, using resistance to geneticin conferred by the KanMX4 marker to score the deletion allele. These crosses also served to generate new spore clones carrying the deletions in each mating type and with different combinations of auxotrophic markers. We used these spore clones to determine the consistency of the survival characteristics in two or more haploid strains for each mutant, as shown in Figures 17, and to construct our own homozygous diploids for testing, as shown in Figures 8 and and99.

Figure 1.
Survival vs. X-ray dose for two haploid dot1 deletion strains and an hht2-K79Q mutant haploid. This mutant (from K. Struhl) is chromosomally deleted for both of the genes that encode histone H3 (HHT1 and HHT2) and both of the histone H4 genes (HHF1 and ...
Figure 7.
Survival vs. X-ray dose for two haploid dcc1 deletion strains. A wild-type and a rad51Δ haploid are included for comparison.
Figure 8.
Survival vs. X-ray dose for diploids homozygous for dot1Δ, mdm20Δ, nat3Δ, vid21Δ, and dcc1Δ mutations. Survival data for a wild-type and a rad51null diploid are included for comparison.
Figure 9.
Survival vs. X-ray dose for diploids homozygous for gcn5Δ, hfi1Δ, spt7Δ, and spt20Δ mutations. Survival data for a wild-type and a rad51null diploid are included for comparison.

Table 1 shows spore-viability data for each of the 11 deletion mutants we chose for further study, crossed with our MATα wild-type strain, g1201-4C. This strain is isogenic in background with the strains used for constructing the deletion library. It was made by crossing the deletion strains carrying multiple auxotrophic markers with the prototrophic strain X2180-1B, which shares the same strain background (see materials and methods). Crosses generated haploid spores in each mating type carrying only the lys2Δ marker (from BY4742) or the met15Δ marker (from BY4741). These single-auxotroph strains can be mated with the deletion mutants created in the BY4741 or BY4742 strains, respectively, and the resulting diploids can be selected on minimal medium. These diploids are heterozygous for five auxotrophic mutations in addition to the deletion of interest. In tetrad analysis these segregating markers, along with mating type, help to identify polyploid or aneuploid cells and false tetrads.

Spore viability and available cosegregation data for nine deletion-mutant heterozygous diploids

Table 1 reveals that in 9 of 11 deletion strains analyzed, meiotic spore viability from the heterozygous diploids was high, and the deletion alleles as scored by geneticin resistance segregated 2+:2− per tetrad, as expected. In eight mutants, the geneticin-resistance phenotype cosegregated with an X-ray-sensitive phenotype that could be readily observed on replica plates given 156 krad of radiation. Fifteen or more tetrads with four viable spores were monitored for cosegregation for each mutant. In the case of the spt7 deletion, a second cross was needed, since poor spore viability led to only three tetrads with four live spore clones in the first cross. Since these three tetrads and other partial tetrads showed convincing segregation for X-ray sensitivity, we hypothesized that the poor viability was unrelated to the deletion. A backcross to wild type using an spt7Δ spore clone from one of these tetrads confirmed this by giving high spore viability and also confirmed the cosegregation relationship, as shown in Table 1. The tetrad data in Table 1 show that an unlinked secondary marker is not responsible for the X-ray sensitivity in these eight mutants, and the high spore viability and 2+:2− segregation of recessive markers from each parent effectively rules out polyploidy. The possibility of a secondary mutation closely linked to the deletion itself is not excluded, but in Saccharomyces the probability of such close linkage of mutations by chance is <1%. We conclude that at least for this genetic background, the deletion allele is both necessary and sufficient to confer the observed X-ray sensitivity in each of the first eight mutants in Table 1. For a ninth mutant, the gcn5 deletion, a minimal X-ray sensitivity that was apparent in survival curves (Figure 3) was insufficient to permit reliable scoring of this phenotype in replica-plated tetrad sets, as discussed below.

Figure 3.
Survival vs. X-ray dose for two gcn5 and two hfi1 haploid deletion strains. A wild-type and a rad51Δ haploid are included for comparison.

Two mutants, those with deletions in the HTL1 and DEF1 genes, failed to behave in the same way as those discussed above. Spore viability from the crosses with wild type was poor in each case (see Table 1). In addition there was wide variation in the size of the germinating spore colonies, with many weak spore clones that could not be reliably scored for IR sensitivity. Aneuploidy or polyploidy is a likely possibility in these crosses, especially in the case of htl1Δ, which is itself known to lead to increased frequencies of spontaneously polyploidized cells (Lanzuolo et al. 2001). Since polyploidy by itself is well known to affect IR sensitivity (Mortimer 1958; Laskowski 1960), it is difficult to assess without reconstructing the strains whether these deletion mutations also confer sensitivity. We have not pursued the htl1Δ and def1Δ mutants further.

X-ray survival curves:

We performed X-ray survival curves for at least two haploids and one homozygous mutant diploid strain for the 9 of 11 deletion mutants in Table 1 that showed 2+:2− segregation for geneticin resistance when crossed to wild type. The results are shown in Figures 19. Additional whole or partial survival assays for many of the mutants (not shown) served to confirm these data. We prefer to present individual survival assays instead of averaging measurements at each dose from separate curves, in part because dose points within a curve are related on the basis of serial dilutions and are not independent measurements. In addition, their accuracy in different curves will vary according to colony count. Hence, taking mean values may be misleading. We include haploid wild-type strains with a genetic background isogenic to the mutants. These are g1201-4C or MW5067-1C, each derived from crosses as described above. These strains were isolated as fresh spore clones from crosses with high viability and from tetrads with four viable spores; hence the presence of any gross chromosomal abnormalities that could have accrued in the parent strain over time is unlikely. A survival curve (not shown) for strain BY4742, the MATα haploid wild type from the deletion collection, was almost identical to that of MW5067-1C. The diploid shown, B4743, is the wild type from the deletion collection.

It can be seen that eight of the mutant strains are significantly more X-ray sensitive than wild type in both haploid and diploid configurations. The ninth mutant, deleted for GCN5, appears to show marginal sensitivity. None of these mutants show such strong sensitivity as the major recombinational repair mutants. These are represented by the rad51null haploid and diploid strains shown in the figures for comparison. Results and discussion for each mutant are detailed below.

DOT1—histone methylation is involved in IR repair:

We find that haploid and homozygous diploid strains deleted for the DOT1 gene (Singer et al. 1998) show significant X-ray sensitivity (see Figures 1 and and8).8). It can be seen that the dot1Δ haploid survival curves are about equal to that of a deletion mutant of the RAD5 gene. This typifies midrange sensitivity that is substantially less than that of the rad51Δ mutant (Figure 1). Interestingly, the homozygous dot1Δ/dot1Δ mutant diploid (Figure 8) is less sensitive compared to the wild-type diploid than the dot1Δ haploid strains are to haploid wild types (Figure 1). This could imply a role in repair of recessive lethal damage, such as base damage rather than double-strand breaks (DSBs), which are thought to be dominant lethal lesions in the absence of repair (reviewed in Game 1983). Alternatively, DOT1 could mediate DSB repair that primarily involves sister chromatids rather than homologous chromosomes. The DOT1 gene is highly conserved throughout eukaryotes (Feng et al. 2002) and its product has a single known function, the methylation of histone H3 protein at one residue, lysine-79, in the core of the protein (Feng et al. 2002; Ng et al. 2002; Van Leeuwen et al. 2002). This methylation is required for DNA silencing near telomeres and elsewhere (Ng et al. 2002, 2003), and dot1 mutants were initially isolated and named on the basis of loss of this function (disruptor of telomeric silencing; Singer et al. 1998). DOT1 is important in differentiating heterochromatin from euchromatin (Ng et al. 2003). To determine whether the DOT1 gene functions in repair via its known activity in histone H3 lysine-79 methylation or via some other unidentified function, we obtained mutants from Kevin Struhl in which the lysine-79 residue in histone H3 is replaced by another amino acid that is not a substrate for methylation (Ng et al. 2002). We expected that such mutants would be similar in IR sensitivity to dot1null mutants even in a DOT1 wild-type background if lysine-79 methylation is required for normal DNA repair.

K. Struhl provided three mutants in which the two chromosomal genes for histone H3 are deleted and a histone H3 gene (on a CEN plasmid) in which lysine-79 is replaced by alanine, proline, or glutamine, respectively, is substituted (Ng et al. 2002). We find that all three of these mutant strains show X-ray sensitivity in spot tests that is similar to that of the dot1Δ mutant. Figure 1 shows a survival curve for a mutant in which lysine-79 is replaced by glutamine. In the same figure, it can be seen that no IR sensitivity is present in a strain (UCC1111) in which a wild-type histone H3 gene is provided on a plasmid to cover the chromosomally deleted histone H3 genes. This confirms that the mutant alleles themselves, rather than the location of the histone H3 gene on a plasmid, are responsible for the IR sensitivity of the H3 lysine-79 replacement mutants. In comparing the hht2-K79Q mutant with the dot1Δ mutant, we are comparing the effect of histone H3 containing a glutamine at residue 79 vs. histone H3 containing an unmethylated lysine at residue 79. While these altered histone proteins may not be exactly equivalent, the fact that both strains are significantly sensitive to ionizing radiation provides strong evidence of a repair function for lysine methylation in the core of histone H3. We are currently determining which repair pathways are affected in the dot1 mutant by constructing and studying dot1 rad double-mutant strains carrying blocks in each of the currently known IR-repair mechanisms.

SPT7, SPT20, HFI1, and GCN5—components of the Spt-Ada-Gcn5-acetyltransferase complex:

Figures 2 and and99 show that deletions of the SPT7 and SPT20 genes each confer modest but consistent IR sensitivity in haploids and detectable sensitivity in diploids. The SPT genes in yeast were identified by Winston et al. (1984) on the basis of a mutant phenotype that involves suppression of the effects of Ty elements inserted into the promoters of other yeast genes. SPT7 and SPT20 code for two components of the yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex (Grant et al. 1997). SAGA is a conserved multiprotein complex involved in normal transcription in yeast. Loss of the complex results in altered mRNA levels for ~10% of the genes in Saccharomyces (reviewed in Wu and Winston 2002). Its components include the catalytic subunit Gcn5p, which is a histone acetyltransferase (Brownell et al. 1996). The histone acetylation activity of Gcn5P is modulated by the associated adaptor proteins Ada2p and Ada3p (Marcus et al. 1994; Horiuchi et al. 1995) and these three proteins also occur as parts of a second complex called ADA (Eberharter et al. 1999). In addition, the SAGA complex interacts with TATA box binding protein and, in the classical form of SAGA that contains the Spt8 protein, inhibits its interaction with the TATA box at some promoters (Sterner et al. 1999; Belotserkovskaya et al. 2000). The Spt7 and Spt20 proteins, together with the product of the HFI1 gene, [also known as ADA1 (Horiuchi et al. 1997) and originally identified as SUP110 (Brown 1994)], are each believed to be essential for the structural integrity of the SAGA complex, which does not form if any of them are absent (Sterner et al. 1999). SAGA includes additional known proteins including the products of TAF genes and can also exist in one or more alternate forms (Sterner and Berger 2000; Wu and Winston 2002).

Figure 2.
Survival vs. X-ray dose for two spt7 and two spt20 haploid deletion strains. A wild-type and a rad51Δ haploid are included for comparison.

Because of the functional relationship of GCN5 and HFI1 with the SPT7 and SPT20 genes, we also undertook genetic crosses to wild type with the gcn5 and hfi1 deletion mutants and performed survival assays of haploid and diploid strains, as with the other mutants. As shown in Figure 3, there is unequivocal X-ray sensitivity for the hfi1 deletion strains. The haploid hfi1 survival curves in Figure 3 show slightly greater sensitivity than the gcn5 curves in the same figure or the spt7Δ and spt20Δ strains in Figure 2. The homozygous diploid hfi1Δ/hfi1Δ strain is very close in survival to the spt7Δ/spt7Δ and spt20Δ/spt20Δ diploids (Figure 9). We found that the IR-sensitivity phenotype of the hfi1Δ mutant could be readily scored in irradiated replica-plate assays and cosegregates with the deletion allele. We also confirmed that the hfi1 deletion strain is auxotrophic for inositol, as expected for this mutant (Horiuchi et al. 1997), and that inositol auxotrophy cosegregates with X-ray sensitivity and with the KanMX4 marker. While this work was in progress, a new mutant (srm12) that was identified in a screen for decreased spontaneous mutagenesis to the mitochondrial rho state was shown to be a nonsense allele of the HFI1 gene and was also shown to confer IR sensitivity (Koltovaya et al. 2003). A diploid homozygous for this mutation showed sensitivity to Co60 gamma rays (Koltovaya et al. 2003) that was comparable to our observations (Figure 9) for the hfi1Δ homozygous mutant diploid treated with X rays.

In contrast to hfi1Δ, the gcn5 deletion confers at most marginal IR sensitivity. The strains whose survival is shown in Figures 3 and and99 were consistently more sensitive than wild type, but the phenotype is sufficiently mild that we were unable to demonstrate that X-ray sensitivity cosegregated with the gcn5 deletion using replica plates of spore clones from meiotic tetrads. We then took 20 spore clones from five meiotic tetrads and measured survival at a single dose (122.9 krad/12.29 Gy) by counting plated colonies, as in our survival curve assays. We found a small difference between mutant and wild-type average survival, but this was largely masked by variation between the individual measurements. The finding that the gcn5Δ mutant is scarcely IR sensitive and certainly less sensitive than the other SAGA mutants we studied may implicate functions other than histone acetylation in mediating the repair functions of this complex. A role for the complex in the transcriptional activation of one or more repair proteins could explain the observation that mutants in genes required for its structural integrity are more sensitive than a mutant deleted only for the histone acetylation activity. Analysis of sensitivity in strains lacking other components of SAGA and its alternate and related complexes will clarify which of their activities is involved in recovery from IR.

NAT3 and MDM20—subunits of the Nat3B protein:

Survival curves for two haploid strains containing the deletion of NAT3 and two strains deleted for MDM20 are shown in Figure 4. Curves for diploid strains homozygous for each of these deletions are shown in Figure 8. The nat3 and mdm20 deletions confer comparable sensitivity that is greater than that of the other mutants described here or by Game et al. (2003). While this work was in progress, it was shown that NAT3 and MDM20 code for subunits of a single protein, NatB Nα-terminal acetyltransferase (Polevoda et al. 2003; see also Singer and Shaw 2003). Hence, the similarity in their IR-sensitive phenotypes is to be expected. The NAT3 gene was previously identified as the probable catalytic subunit of this protein (Polevoda et al. 1999), which is one of three known enzyme complexes that between them acetylate the N termini of a large number of yeast proteins. Specifically, the NatB protein acetylates yeast proteins with N termini consisting of Met-Glu or Met-Asp and subclasses of proteins with Met-Asn or Met-Met and partially acetylates some proteins with other termini (see Polevoda and Sherman 2003).

Figure 4.
Survival vs. X-ray dose for two mdm20 and two nat3 haploid deletion strains. g1229-1D mdm20Δ is unable to grow at 37° and g1229-9B mdm20Δ can grow at 37°; see text. A wild-type and a rad51Δ haploid are included ...

Previously reported phenotypes for both nat3 and mdm20 deletion strains include increased sensitivity in spot tests to several chemicals that lead to DNA damage, including camptothecin, bleomycin, hydroxyurea, and caffeine (Polevoda et al. 2003), an inability to grow at 37°, reduced growth on nonfermentable carbon sources, diminished mating efficiency, and other effects confirmed and reviewed by Polevoda et al. (2003). In our hands, at 30° radiation sensitivity convincingly cosegregated 2+:2− with the deletion alleles, for both mdm20 and nat3 deletion strains when crossed with wild type (see Table 1). Each mutant also showed a temperature-conditional growth phenotype. However, as reported elsewhere (Singer and Shaw 2003), this phenotype was more pronounced in mdm20Δ strains, which showed essentially no growth at 37°, than in nat3Δ strains, which we found to show limited but real growth at 37° over a period of 2 or more days.

In the cross of the original temperature-sensitive mdm20Δ mutant to our wild-type strain g1201-4C, a fraction of the tetrads showed a digenic segregation for temperature-sensitive growth, such that one or in some cases both the spore clones containing the mdm20Δ::KanMX4 allele showed wild-type growth at 37°. None of the MDM20+ spores were inviable at 37°, indicating that the deletion was necessary but not always sufficient to confer the temperature-conditional growth phenotype in this cross and that an unlinked suppressor of the conditional lethality was also segregating in some tetrads. We mated mdm20Δ spore clones that were able to grow at 37° with those that were inviable at this temperature. The resulting diploids were viable at 37°, indicating that the suppressor phenotype is dominant. However, radiation sensitivity was apparently unaffected by this suppressor, since mdm20Δ spore clones that were viable at 37° remained IR sensitive both at this temperature and at 30°. Survival curves (at 30°) of a temperature-conditional mdm20Δ strain (g1229-1D) and one able to grow at 37° (g1229-9B) were equivalent (Figure 4).

We observed a similar phenomenon with nat3Δ strains. In this case, about half the nat3Δ spores showed a growth defect at 37° that was more pronounced than that of the initial nat3Δ mutant culture, although still not as strong as that seen in mdm20Δ strains. We inferred that a weak suppressor of the temperature-conditional nat3Δ phenotype was present in our starting culture. We tested whether this influenced IR sensitivity by comparing survival of a strongly temperature-conditional spore clone with that of a weakly temperature-conditional one. We also tested the effect of temperature on IR sensitivity in each strain by incubating parallel sets of plates at 25°, 30°, and 37° immediately after irradiation. (Growth prior to irradiation in this experiment was at 25°.) Even the strain with the stronger temperature-sensitive growth defect was able to form small but countable colonies at 37°. The results are presented in Figure 5, where it can be seen that there is a small effect of temperature on survival in both strains, but no clear difference between the strains. There is also a minor difference in the survival curves at two temperatures in the wild-type strain (Figure 5). Although this is less than that seen in the two nat3Δ strains, it seems likely that as with mdm20Δ, the IR-sensitive phenotype and the temperature-sensitive growth defect in the nat3 deletion are conferred through separate mechanisms.

Figure 5.
Survival vs. X-ray dose for nat3Δ and wild-type strains pregrown at 25° and incubated at 25° or 37° after X-ray treatment. Survival data for a rad51null haploid pregrown and incubated at 30° are included for comparison. ...

These observations are consistent with findings of Singer et al. (2000), who identified and studied nine dominant suppressor mutations of the mdm20 deletion. They found that all nine of these suppressors represent missense alleles in the structural genes for actin or tropomyosin (ACT1 and TPM1, respectively). They argued from this and other evidence that many of the phenotypes of the mdm20 deletion mutant arise from its destabilization of actin-tropomyosin interactions, causing partial loss of function. It has been shown independently that the NatB complex is required for acetylation of both actin (Polevoda et al. 1999) and tropomyosin (Singer and Shaw 2003), supporting this hypothesis. However, Polevoda et al. (2003) point out that several of the many other potential target proteins for the NatB acetyltransferase are involved in repair. In addition, as in the case of the mdm20 deletion (Singer et al. 2000), they identified suppressors of the nat3Δ temperature-sensitive phenotype that represented alterations in the ACT1 and TPM1 genes (Polevoda et al. 2003). In spot tests, these suppressors did not suppress the sensitivity of nat3Δ mutants to hydroxyurea and camptothecin, although the TPM1 (but not ACT1) mutants did partially suppress sensitivity of nat3Δ strains to bleomycin (Polevoda et al. 2003). These authors concluded that the increased sensitivity of nat3Δ and mdm20Δ strains to DNA-damaging agents in spot tests may arise from effects of the mutations on other target proteins. If so, this could explain our observation that suppressors of the temperature-conditional phenotype of the mdm20Δ and nat3Δ mutants fail to affect their radiation-sensitive phenotypes. Alternatively, partial restoration or bypass of NatB function by the suppressor might be sufficient for growth but inadequate for IR repair. Additional studies of missense mutants involving potential NatB target enzymes that cannot be acetylated and of mdm20 and nat3 double-mutant combinations with these and other mutants should clarify the cause of the IR sensitivity conferred by these deletions.


The name VID21 was standardized for ORF YDR359C by the Saccharomyces Genome Database in August 2003. The name represents a suggested function in vacuolar import and degradation, proposed earlier for 20 other genes numbered VID1VID20 (Hoffman and Chiang 1996). Very recently YDR359C has also been shown to be the ORF for an independently named gene, EAF1, identified from a protein (Esa1-associated factor 1) that is a member of the NuA4 histone acetyltransferase complex (Doyon et al. 2004). Strains deleted for VID21 were reported by Bennett et al. (2001) to be IR sensitive. We observed significant IR sensitivity in the BY4742 MATα strain deleted for ORF YDR359C (VID21/EAF1) and confirmed that an IR-sensitive phenotype cosegregates with the deletion allele in meiotic tetrads (see Table 1). X-ray survival curves for one homozygous diploid and two haploid vid21Δ::KanMX4 mutant strains are shown in Figures 6 and and8,8, respectively. It can be seen that the deletion confers significant X-ray sensitivity in both haploids and in the homozygous diploid strain. The mutant effect is only slightly less than that shown by the mdm20 and nat3 deletion strains in Figure 4.

Figure 6.
Survival vs. X-ray dose for two haploid vid21 (ORF YDR359C) deletion strains. A wild-type and a rad51Δ haploid are included for comparison.

The recent finding that VID21/EAF1 encodes a member of the NuA4 HAT complex (Doyon et al. 2004) will help to clarify its role in repair. The catalytic unit of this complex is the essential gene ESA1, whose product acetylates in vivo up to four lysine residues in the tail region of histone H4 and probably some sites on other histone proteins to a lesser degree (Allard et al. 1999; Sterner and Berger 2000). Missense mutants that are viable but partially defective in the activity of Esa1 protein and histone H4 mutants in which the target lysine residues are altered to nonacetylated amino acids have both been shown to confer sensitivity to MMS and camptothecin (Bird et al. 2002). Another mutant involving the NuA4 HAT complex, yng2null, has also been shown to be sensitive to MMS and camptothecin and to show a reduction in the repair of MMS-induced DSBs in an assay using pulsed-field gel electrophoresis (Choy and Kron 2002). It has been suggested (Bird et al. 2002) that some NuA4 complex mutants may affect replication-coupled repair, on the basis of their increased sensitivity to camptothecin, which is a topoisomerase I poison known to lead to DSBs during replication (D'Arpa et al. 1990).


The DCC1 gene is represented by ORF YCL016C. Its gene product was identified as a protein that binds to others involved in sister-chromatid cohesion, and the gene was named DCC1 on the basis of its mutant phenotype of defective sister-chromatid cohesion (Mayer et al. 2001). A human homolog, hDCC1, has been cloned and characterized (Merkle et al. 2003). Strains from the genomic deletion library that were deleted for YCL016C (DCC1) were reported as IR sensitive in spot tests by Bennett et al. (2001), and we verified that this phenotype is conferred by the deletion (see Table 1). Detailed survival curves (Figure 7) confirm this sensitivity, with two haploid spore clones showing excellent agreement. A diploid homozygous for the dcc1Δ::KanMX4 deletion, constructed by mating two spore clones, also shows substantial X-ray sensitivity, as shown in Figure 8.

It is likely that recombinational repair involving sister chromatids could be defective in dcc1 deletion strains given the known role of DCC1 in enabling sister-chromatid cohesion (Mayer et al. 2001). Recombinational repair using sister chromatids as templates is believed to be a major route for repair of IR-induced DSBs in wild-type haploid yeast cells (see Kupiec 2000; Game 2000 for reviews). In addition, work with several organisms, including Saccharomyces (Sjogren and Nasmyth 2001), Schizosaccharomyces (Hartsuiker et al. 2001), and Coprinus (Cummings et al. 2002), has shown that mutants with defects in sister-chromatid cohesion are often sensitive to radiation and compromised in recombinational repair (reviewed by Strunnikov and Jessberger 1999). Dcc1p forms part of an “alternative” replication factor C (RFC) complex (Mayer et al. 2001) that has recently been shown to load the sliding-clamp proliferating cell nuclear antigen (PCNA) onto DNA (Bermudez et al. 2003). The same clamp-protein (PCNA) is also loaded onto DNA by the related RFC1 complex, which contains several proteins in common but lacks Dcc1p (Cullmann et al. 1995; reviewed in Waga and Stillman 1998). It has been proposed that the Dcc1p-containing complex loads PCNA onto DNA at certain sites and that this effects a change in the replication-polymerase machinery to enable cohesin proteins to be laid down at these sites, leading to sister-chromatid cohesion (Mayer et al. 2001). In addition, modified forms of PCNA are major players in controlling postreplication/translesion synthesis repair. Modification of PCNA is mediated by the RAD6, RAD18, RAD5, and UBC13 gene products in a complex way to provide ubiquitinated and SUMO-ylated forms of this protein, and these modifications are thought to control its roles in different repair mechanisms (reviewed in Matunis 2002; see also Hoege et al. 2002; Stelter and Ulrich 2003).

Six other proteins are in the same heptameric alternative RFC complex that includes the Dcc1 protein. These consist of four RFC proteins that are essential and occur in the other RFC complexes, plus the products of the CTF8 and CTF18 genes (Hanna et al. 2001; Mayer et al. 2001; Naiki et al. 2001). We have not yet tested strains deleted for either CTF8 or CTF18, but note that Bennett et al. (2001) have reported that strains deleted for CTF8 showed IR sensitivity in spot tests. CTF18 was initially identified and named CHL12 on the basis of a screen for mutants with increased chromosome loss (Kouprina et al. 1993). The deletion mutant was found to show elevated spontaneous mitotic recombination, slow growth, and cold-sensitive phenotypes (Kouprina et al. 1994).

Testing the mutants for cross-sensitivity to ultraviolet radiation:

Yeast mutants that are sensitive to IR frequently show cross-sensitivity to ultraviolet radiation. To determine if the IR-sensitive mutants discussed above also confer UV sensitivity, we obtained UV survival curves for at least one strain carrying each mutant. Results are shown in Figure 10, which includes the wild-type strain presented above and a rad14null mutant defective in excision repair as a known highly UV-sensitive mutant control. It can be seen that none of the nine other mutants confer high UV sensitivity, and all except possibly the mdm20Δ and nat3Δ strains fall within or very close to the wild-type range. In the case of nat3Δ and mdm20Δ, we also studied meiotic tetrads from heterozygous diploids to test for segregation of a UV-sensitive phenotype. On UV-irradiated replica plates, we could not observe any clear segregation for a sensitivity phenotype among spore clones from five and 13 complete tetrads segregating for mdm20Δ and nat3Δ, respectively. The weaker growth of the mutant spore clones tended to complicate the interpretation, but we inferred that the difference in sensitivity between wild-type and mutant spore clones was at most minor.

Figure 10.
Survival vs. ultraviolet radiation dose for 10 haploid deletion mutant strains compared with wild type. A rad14null mutant, defective in nucleotide excision repair, is included for comparison and shows the high sensitivity expected for such strains.


We have documented here an IR-sensitive phenotype for haploid and homozygous diploid yeast mutants involving deletions of the DOT1, SPT7, SPT20, HFI1, MDM20, NAT3, VID21 (EAF1), and DCC1 genes. We have found at most a borderline X-ray sensitivity in mutants deleted for another gene, GCN5. In each case except GCN5, we have demonstrated that IR sensitivity cosegregates with the deletion allele in meiotic tetrads. To confirm that a deletion mutation confers IR sensitivity, we consider it essential to obtain quantitative survival data and also to demonstrate that the phenotype is conferred by the deletion itself. With this in mind, we confirmed that the nat3Δ, vid21Δ, and dcc1Δ mutations do confer IR sensitivity, following a previous report (Bennett et al. 2001) that identified strains containing these deletions as sensitive in qualitative tests. Strains reported as sensitive that carried deletions in htl1Δ or def1Δ/vid31Δ (Bennett et al. 2001), gave very poor spore viability in crosses of the MATα parents to wild type. We did not confirm that sensitivity is conferred by the deletion rather than another change such as altered ploidy, although both factors could have contributed to the sensitive phenotype. Apart from these five mutants, we focused our initial screen on mutants that were missing from the original collection screened by Bennett et al. (2001). Among this group, we focused on those that were either absent from the Brown laboratory pool or too low in preirradiation hybridization signal to be assessed properly in their screen. In addition, we screened some mutants that were absent from the Bennett et al. (2001) screen, but had a low rank in the Brown pool screen (suggesting sensitivity) and had not been individually tested. Finally, we screened deletions involving three genes because their products formed complexes with the product of the SPT20 gene whose deletion we identified as IR sensitive.

The results reported here complement the earlier identification of the RAD61 gene, which resulted from information from the pool assay (Birrell et al. 2001; Game et al. 2003) and has recently been reported to show a defect in sister-chromatid cohesion (Warren et al. 2004). We are continuing to characterize both the rad61 deletion and the mutants reported on here in terms of epistasis relationships and phenotypes involving mutation and recombination. No inferences can be drawn about the overall frequency of genes involved in IR sensitivity from this work, because we used nonrandom criteria in choosing the initial set to study.

We find that none of the mutants reported on here confers substantial cross-sensitivity to UV radiation. This may argue against a significant role in postreplication repair (PRR) for these mutants. While not all known PRR mutants are significantly radiation sensitive, those that do confer IR sensitivity, such as rad6null, rad18null, and rad5null, usually also confer substantial UV sensitivity. In contrast, mutants in recombinational repair usually confer only minor UV sensitivity (see Game 1983).

We searched the online data sets at the Saccharomyces Genome Database to determine if the wild-type genes whose mutants we studied are induced by radiation. We found no evidence in the literature for a strong and consistent induction of any of these genes by IR. This is not necessarily surprising in view of reports that there is a poor correlation between Saccharomyces genes that are induced by toxic agents and genes responsible for resisting those agents (Begley et al. 2002; Birrell et al. 2002).

Of the 9 mutants we report on here perhaps the most interesting is the dot1 deletion, which specifically eliminates the methylation of the lysine-79 residue in the core of the histone H3 protein (Feng et al. 2002; Ng et al. 2002; Van Leeuwen et al. 2002). Histone H3 mutants (supplied by K. Struhl) in which this target lysine is replaced by other amino acids show nearly equivalent IR sensitivity (see Figure 1). This provides strong evidence that this methylation, which cannot occur on the substituted amino acids, is responsible for the role of the highly conserved DOT1 gene in IR resistance. It links histone modification to DNA repair and provides an opportunity to use epistasis analysis and additional characterization to identify the repair pathways involved.

In addition to histone H3 lysine-79 methylation, we have shown that five genes involved in histone acetylation and transcriptional regulation (HAT) complexes play some role in IR resistance. Four of these, SPT7, SPT20, HFI1, and GCN5, encode components of the SAGA protein complex, while VID21/EAF1 encodes a protein in the separate NuA4 HAT complex. Given the milder phenotype in gcn5Δ compared to the other HAT mutants, it is plausible that the IR sensitivity seen in these different HAT complex mutants could be mediated indirectly by effects on transcriptional activation, which could influence the activity levels of other repair genes, rather than via histone acetylation per se. This is also possible in the case of the dot1null mutant, but perhaps less likely given the greater degree of IR sensitivity and the known role of DOT1 in the control of gene silencing and chromatin structure. Two other genes among the nine, NAT3 and MDM20, are also involved in acetylation, although there are multiple target protein(s) for the NatB acetylase that they encode (Polevoda and Sherman 2003). In contrast, it seems probable that the deletion of DCC1 leads to X-ray sensitivity through disruption of sister-chromatid cohesion, since the gene product has a known function in ensuring this process (Mayer et al. 2001), and it is known that mutants affected in sister-chromatid cohesion can be sensitive to radiation and compromised in recombinational repair (reviewed by Strunnikov and Jessberger 1999).

The diverse roles of the genes studied here confirm that much remains to be learned about recovery from IR damage. None of them has a product that is known to interact directly with DNA. Genes whose products modify other proteins that themselves may interact with DNA are emerging as a significant class of all those involved in IR recovery. Such genes include the well-known RAD5, RAD6, and RAD18 loci (Hoege et al. 2002; Stelter and Ulrich 2003) as well as some of those more recently identified. Since some protein-modifying enzymes such as the Nat3 complex have multiple targets, knowledge of their enzymatic function does not necessarily clarify their role in repair or even which repair mode they are involved in. However, double-mutant analysis can take advantage of the many Saccharomyces genes already associated with identified repair pathways. This should determine which if any of the currently known pathways require the functions recently identified as relevant.


We thank J. Martin Brown and James A. Brown at Stanford for generously sharing strains and information. We thank Kevin Struhl for providing mutant strains with substitutions at the K79 residue of the histone H3 protein. This work was supported by National Institutes of Health grant GM-5997901 to J.C.G.


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