Topoisomerase I plays a vital role in relieving tension on DNA strands generated during replication. However if trapped by camptothecin or other DNA damage, topoisomerase protein complexes may stall replication forks producing DNA double-strand breaks (DSBs). Previous work has demonstrated that two structure-specific nucleases, Rad1 and Mus81, protect cells from camptothecin toxicity. In this study, we used a yeast deletion pool to identify genes that are important for growth in the presence of camptothecin. In addition to genes involved in DSB repair and recombination, we identified four genes with known or implicated nuclease activity, SLX1, SLX4, SAE2, and RAD27, that were also important for protection against camptothecin. Genetic analysis revealed that the flap endonucleases Slx4 and Sae2 represent new pathways parallel to Tdp1, Rad1, and Mus81 that protect cells from camptothecin toxicity. We show further that the function of Sae2 is likely due to its interaction with the endonuclease Mre11 and that the latter acts on an independent branch to repair camptothecin-induced damage. These results suggest that Mre11 (with Sae2) and Slx4 represent two new structure-specific endonucleases that protect cells from trapped topoisomerase by removing topoisomerase-DNA adducts.
DNA topoisomerase I (Top1) is an essential enzyme that relaxes DNA supercoiling ahead of the replication fork by transiently cutting and religating a single strand of the DNA double helix. This reaction involves forming a covalent (3′-phosphotyrosyl)-enzyme-DNA complex. These Top1-DNA covalent complexes are normally transient, but the anticancer drug camptothecin stabilizes this complex by slowing the rate of DNA religation (Svejstrup et al. 1991). This camptothecin-induced stabilized complex can then lead to a DNA double-strand break and cytotoxicity if it blocks a replication fork (Hsiang et al. 1989). In addition, various other types of DNA damage, such as base mismatches and abasic lesions, have been shown to stabilize the Top1-DNA complex (Pourquier et al. 1999).
The lesion created by the collision of the replication fork with the Top1-DNA complex requires specialized enzymes for its repair since the enzyme is covalently bound to the 3′ end of the break and must be removed before the DNA can be religated. In the budding yeast Saccharomyces cerevisiae, an enzyme, tyrosyl-DNA phophodiesterase (Tdp1), has been found that can specifically hydrolyze this lesion (Yang et al. 1996; Pouliot et al. 1999). However, yeast mutants deleted in TDP1 show little or no sensitivity to camptothecin (Vance and Wilson 2002) and it has been shown that the structure-specific heterodimer endonuclease Rad1 Rad10 functions as a redundant pathway for removing the Top1-DNA lesion (Vance and Wilson 2002). Thus, whereas the tdp1 and rad1 deletion mutants have little or no sensitivity to camptothecin, the double mutant is hypersensitive to the drug (Vance and Wilson 2002). In addition, deletion of the endonuclease-encoding gene MUS81 confers sensitivity to camptothecin apparently through a pathway parallel to Tdp1 and Rad1, as the triple mutant tdp1 rad1 mus81 is more sensitive than the double-mutant tdp1 rad1 (Liu et al. 2002; Vance and Wilson 2002).
It would appear, however, that still more enzymes may be involved in the repair of the Top1-DNA complex, as mutants in RAD52, which abolish double-strand break repair, are considerably more sensitive to camptothecin than are the double mutation tdp1 rad1 (Vance and Wilson 2002) and the triple mutant mus81 rad1 tdp1 (Liu et al. 2002). To identify any such alternative pathways, we performed a genome-wide screen to detect all nonessential genes that are important for protection from growth inhibition or killing produced by continuous exposure to camptothecin. The recent completion of a systematic deletion of all open reading frames in yeast (Giaever et al. 2002) has provided a powerful new tool for screening for genes whose deletion produces sensitivity to cytotoxic drugs. We and others have used this collection of mutants to identify novel genes whose deletion confers sensitivity to ultraviolet radiation (Birrell et al. 2001), ionizing radiation (Bennett et al. 2001; Game et al. 2003), and other DNA-damaging agents (Begley et al. 2002; Chang et al. 2002; Wu et al. 2004). One of the advantages of this resource is that the gene replacement cassette contains two molecular “bar code” tags or unique 20-base oligonucleotide sequences, which allow for unique identification of the strain in a pool of all deletion mutants by PCR amplification of the tags and subsequent hybridization to a high-density oligonucleotide array containing the corresponding complementary sequences (Giaever et al. 2002).
From this screen we identified two further pathways involved in repairing the Top1-DNA complex, one involving Slx4 (but not its partner Slx1), which acts in a parallel pathway to Tdp1 and Rad1/Rad10, and Sae2, which appears to act by stimulating the endonuclease activity of Mre11.
MATERIALS AND METHODS
Yeast strains, deletion pool, and drug treatment:
Genotypes of the parental diploid yeast strain BY4743, construction of the homozygous diploid deletion strains, and construction of the homozygous diploid deletion pool have been described previously (Giaever et al. 2002). All completed deletion strains are available through Open Biosystems (Huntsville, AL) or EUROSCARF (Frankfurt, Germany). We used a mutant pool of 4728 nonessential homozygous diploid deletion strains. Use of the diploid pool of deletion strains minimizes the possible influence of mutations in other genes produced in the production of the haploid deletion stains.
An aliquot of the pool containing ∼107 cells was diluted 10-fold in YPD and then grown for 6–7 hr. Aliquots were then either mock treated or treated with 50 μm camptothecin for 16 hr and periodically diluted to ensure that the cells remained in logarithmic growth. Following the 16-hr exposure the cells were harvested, genomic DNA extracted, PCR amplified, and hybridized to custom-made Tag3 gene chips (Affymetrix, Santa Clara, CA) as described previously (Birrell et al. 2002). Each deletion strain is associated with four hybridization signals on the high-density oligonucleotide array generated in two separate PCR labeling reactions: UPTAG (sense and antisense) and DNTAG (sense and antisense). Equal numbers of cells were harvested in both the control and the treated pools to produce equal pool label intensities. We calculated the background intensity of each array and normalized the data generated in the experimental array to that of the control array to eliminate any bias created during the PCR amplification reaction as described earlier (Wu et al. 2004). An experimental/control intensity ratio was included in the data analysis only if the signal generated in the untreated control array was at least twice the background signal. This limit was chosen so that a maximum value of 0.5 for the ratio of treated/control hybridizations would be obtained if the treated signal was at the background level. In addition, each treated tag that fell within two standard deviations of the background was flagged, indicating that the measured value may be an overestimation of the true ratio (i.e., the true value would likely be lower, indicating greater sensitivity). To yield a more stable estimate of the average treated/control ratio, we averaged the logs of the treated/control ratios for each of the four tags assigned to an individual strain. Those strains that failed to have at least two of the tags significantly above background were not called for an individual experiment. In addition, only those strains that were called in at least two of the three replicate experiments were included in further analysis to determine the ranking of strain sensitivities shown in Table 1. Application of these quality control criteria eliminated 6.3% of all the strains in the pool (i.e., we are working with 4424 of the total 4728 deletion strains). Among the strains of importance to this work that were “not called” in the hybridization experiments because of their low abundance in the pool was the mre11 deletion mutant.
Testing of individual strains for sensitivity to camptothecin:
All individual strains tested were haploids in the BY4741 and BY4742 backgrounds and were generated by the Saccharomyces Genome Deletion Project unless otherwise specified. The double- and triple-deletion strains were the products of tetrad dissection. All the deletions were confirmed by genomic PCR using primer sets flanking the ORF as well as primers within the deleted coding region or the Kanamycin replacement marker. In the case of the slx1 tdp1 double deletion we transformed the tdp1 mutant with an slx1::KanMX cassette amplified by PCR because SLX1 and TDP1 are tightly linked and extremely difficult to obtain by tetrad dissection. This transformant was then backcrossed with a wild-type strain to minimize influence of random mutations introduced during transformation. Spores were selected and confirmed by PCR to be deleted in both SLX1 and TDP1. The mre11-H125N mutants were kindly provided by Lorraine S. Symington (Moreau et al. 1999). Table 1 shows the genotypes of all the strains used.
Measurement of sensitivity to camptothecin was performed according to the published protocol of Vance and Wilson (2002). Briefly, cells were grown to midlog phase in the presence of 1% DMSO and then diluted 1000- to 2000-fold and treated with camptothecin at various concentrations. OD600 was measured for all testing concentrations when the untreated culture of a test strain had proliferated 10 cycles. We then calculated the slope of a plot of ln (fold of OD600 increase) vs. the number of the doubling of the untreated culture. The sensitivity was determined as the ratio of slope of the treated sample divided by that of the untreated sample.
Genomic screen for genes whose deletion produces sensitivity to camptothecin:
To determine which genes when deleted cause sensitivity to continuous exposure to camptothecin, we treated the pool of 4728 deletion strains composing the yeast deletion pool with 50 μm camptothecin for 16 hr and then compared the abundance of the strains in the treated pool with those in a mock-treated pool. This assay measures overall growth inhibition, which integrates slowing of growth and cell killing. Table 2 lists the top 30 sensitive deletion strains and supplemental Table 3 (see supplemental data at http://www.supplemental.data.html) lists the ranking of all 4728 strains to the camptothecin exposure). For convenience we have also listed in Table 2 the rankings of the deletion strains used in this investigation. Not surprisingly, the most sensitive strains were deleted for genes required for recombinational repair, consistent with the fact that camptothecin causes DNA double-strand breaks. In addition to these highly sensitive strains we noted that the deletion of Tdp1 caused slight sensitivity to camptothecin with a ranking of 149 among the 4728 yeast strains. This slight sensitivity is consistent with the existence of other repair mechanisms for removing Top1-DNA complexes in the absence of Tdp1 (Liu et al. 2002; Vance and Wilson 2002). To test our hypothesis that there may be other endonucleases capable of removing the Top1-DNA complex in the absence of Tdp1, we examined the results of the deletion pool screen for other endonucleases that also cause a slight to moderate sensitivity to camptothecin when deleted (defined as in the top 150 sensitive strains to prolonged camptothecin exposure). Four such genes were found: SLX1, SLX4, RAD27, and SAE2. Experiments to determine their phenotype in relation to sensitivity to camptothecin are described below.
Slx4 but not Slx1 is involved in repairing camptothecin-induced damage in the absence of Tdp1:
SLX1 and SLX4 were originally identified as genes synthetically lethal with mutations in SGS1 or TOP3 (Mullen et al. 2001). Slx1 and Slx4 coimmunoprecipitate, and in vitro evidence shows that the proteins form a heterodimeric complex that has strong endonuclease activity (Fricke and Brill 2003). The complex is active in vitro on branched DNA substrates including simple Y, 5′ flap, 3′ flap, replication forks, and Holliday junction substrates, with a preference for the 5′ flap, simple Y, and replication fork structures. Since Rad1/Rad10 and Mus81/Mms4, two endonucleases that preferentially cut a simple Y and 3′ flap structures, respectively (Bastin-Shanower et al. 2003), have both been shown to be required to remove Top1 complexes from DNA, we reasoned that Slx1/Slx4 may have a similar function in removing covalently complexed Top1. We employed an assay developed by Vance and Wilson (2002) that measures growth inhibition during continuous drug exposure to measure the sensitivity of yeast to camptothecin. While the tdp1 and slx4 single-mutant strains were only slightly inhibited by camptothecin, the tdp1 slx4 double mutant was substantially more sensitive to camptothecin than was either single mutant (Figure 1a). This synergistic inhibition by camptothecin is similar to that previously observed for the rad1 and tdp1 deletion mutants (Vance and Wilson 2002), suggesting that Slx4 may represent another pathway to remove Top1 complexes stabilized by camptothecin in the absence of Tdp1. Surprisingly, Slx1 appears not to have the same function as Slx4, as neither slx1 nor the slx1 tdp1 double mutant was more sensitive to camptothecin than was the wild type (Figure 1b). This result provides evidence that Slx1 and Slx4 have separate functions in addition to their shared endonuclease activities (Fricke and Brill 2003).
We next examined the relationship between Slx4, Rad1, and Mus81. The rad1 tdp1 double mutant was more sensitive to camptothecin than was the slx4 tdp1 mutant (Figure 2a), suggesting that Rad1 plays a more active role than Slx4 in the removal of Top1 complexes. As expected, both pathways are engaged only in the absence of Tdp1, as rad1 and slx4 single and double mutants had sensitivities very similar to that of wild-type cells (Figure 2b). However, deletion of SLX4 in the rad1 tdp1 or mus81 tdp1 strains caused additional sensitivity to camptothecin, suggesting that SLX4 is not in the same epistasis group as either MUS81 or RAD1 (Figure 2, a and c). This provides evidence that Slx4 is in another pathway for repair of Top1-DNA complexes.
Sae2 and Mre11 participate in repairing camptothecin-induced damage:
Sae2 was originally identified by its requirement in meiotic recombination, a requirement that can be bypassed by a mutation in SPO11 (McKee and Kleckner 1997; Prinz et al. 1997). In the sae2 mutant, meiotic recombination is blocked at an intermediate stage, and covalent protein complexes are found at the broken ends of double-strand breaks (Keeney and Kleckner 1995). This and other phenotypes of sae2, including repair of mitotic double-strand breaks, are also observed in the separation-of-function mutant mre11s-H125N (McKee and Kleckner 1997; Prinz et al. 1997; Rattray et al. 2001). Mre11 is an endonuclease that forms a complex with Rad50 and Xrs2 to process double-strand breaks during meiosis, and the H125N mutation is in the endonuclease domain (Usui et al. 1998; Moreau et al. 1999). In another separation of function mutant, rad50s-K81I, the covalent protein complex at the DNA ends is Spo11, a topoisomerase II-like protein that initiates meiotic recombination by creating double-strand breaks (Keeney et al. 1997). On the basis of the similarity of the phenotypes of sae2 and rad50s-K81I and mre11s-H125N, it has been suggested that Sae2 is required for the endonuclease activity of the Mre11-Rad50-Xrs2 complex, although no direct biochemical evidence has been reported (Rattray et al. 2001). We therefore asked whether Sae2 is involved in repairing camptothecin-induced DNA damage. As shown in Table 2, the sae2 deletion strain is among the strains most sensitive to camptothecin in the deletion pool.
To examine the relationship between Sae2 and that of the other proteins involved in repairing Top1-DNA complexes we determined the sensitivity of strains with single and double deletions in SAE2 and the other genes in the pathway. Figure 3a shows that the sae2 deletion strain was indeed very sensitive to camptothecin. Surprisingly, deletion of TDP1 in this strain did not cause significantly higher sensitivity. This pattern is similar to that observed for mus81 (Liu et al. 2002; Vance and Wilson 2002). To determine whether Sae2 constitutes an additional pathway to remove Top1 complexes, we studied the genetic relationship between Sae2, Rad1, and Mus81. Figure 3, b and c, shows that deletion of SAE2 in the rad1tdp1 and mus81tdp1 backgrounds caused greater sensitivity to camptothecin than any double-deletion combinations. These results suggest that Sae2 acts in a redundant pathway to protect camptothecin toxicity in the absence of repair by Tdp1, Mus81, or Rad1.
Because of the close similarity of sae2 and mre11s-H125N mutants, we next examined whether Mre11 endonuclease activity is involved in removing Top1. Figure 4a shows that the H125N mutation of MRE11 alone caused a mild sensitivity to camptothecin. Moreover, deletion of TDP1 and the mre11-H125N mutation were synergistic in their sensitivity to camptothecin. This result suggests that Mre11 endonuclease activity is involved specifically in removing Top1 protein complex stabilized by camptothecin at the DNA ends. Deletion of RAD1 in the mre11-H125N tdp1 double mutant caused further sensitivity to camptothecin (Figure 4a), suggesting that Mre11 is in a parallel pathway in the removal of Top1.
The suggestion that Sae2 is required for the endonuclease activity of the Mre11-Rad50-Xrs2 complex (Rattray et al. 2001) predicts that these two mutants would be epistatic. We therefore tested the double mutant sae2 mre11-H125N and, in agreement with this prediction, found that it was no more sensitive than the sae2 deletion strain (Figure 4b). This result is confirmed by our finding that the double-deletion mutant sae2 mre11 has the same sensitivity as the single-deletion mutant mre11 strain (Figure 5b). To test the specificity of the Sae2 for lesions created by Top1, we tested the effect of deletion of SAE2 in a strain deleted in TOP1. As expected, deletion of TOP1 abrogated the sensitivity of wild-type cells to camptothecin, but, more importantly, reversed the sensitivity of the sae2 deletion strain to the drug (Figure 5a). This result demonstrates that Sae2 acts on lesions created by topoisomerase I. As a further test of the repair pathway of Sae2, we crossed strains with deletions in SAE2 and RAD9 and found that their sensitivities to camptothecin were approximately additive (Figure 5c), demonstrating that the protein products of these two genes are acting in different repair pathways (as demonstrated earlier for Tdp1 and Rad9) (Pouliot et al. 1999).
We noted that deletion of SAE2 was previously reported to produce little sensitivity to camptothecin, either by itself or in combination with tdp1 (Liu et al. 2002). We suggest that this discrepancy may be due to the higher sensitivity in the assay we used, which includes growth inhibition in addition to cell killing.
Function of Rad27 in repairing Top1 lesions:
Rad27 is also a flap endonuclease and is required for processing lagging strand Okazaki fragments (Xie et al. 2001). Deletion of RAD27 causes mild sensitivity to camptothecin, with a sensitivity ranking of 82 among the 4742 deletion strains. However, deletion of TDP1 in the rad27 strain did not cause significantly higher sensitivity, consistent with a previous report (Vance and Wilson 2002). Therefore Rad27 is not specifically involved in removing Top1 complexes from DNA ends.
We report in this work the identification of two new pathways, involving the proteins Mre11 and Slx4, for the removal of Top1 from DNA ends. The data have been obtained using genetic analysis of the response of mutant strains of yeast to the Top1 poison camptothecin. This drug provides a useful way of “freezing” the covalent linkage of Top1 to DNA, thereby revealing the enzymes involved in normal resolution of the covalent complex on single-stranded DNA. Previous work of others has identified the tyrosyl-DNA phosphodiesterase Tdp1 and the structure-specific endonuclease Rad1-Rad10 as important in repairing the Top1 DNA covalent complex (Liu et al. 2002; Vance and Wilson 2002). The Mus81-Mms4 endonuclease complex has also been identified as playing a role in protection of cells against camptothecin, either by restarting replication forks that have stalled at Top1 complexes or by directly resecting the DNA ends with covalently attached Top1 from a duplex flap (Vance and Wilson 2002). Our genetic evidence suggests that Mre11 and Slx4 also protect cells against camptothecin and indicates that they are in different branches than the Rad1 and Mus81 pathways. It therefore seems likely that there are three mechanisms to protect cells from Top1-DNA complexes stabilized by camptothecin (Figure 6). First, Top1 can spontaneously cut and religate the DNA. The stalled replication forks then may be restarted by Mus81 and possibly Sgs1 (Kaliraman et al. 2001). If spontaneous reversion of Top1 does not occur, then Tdp1 can free Top1, thereby leaving a 3′ end phosphate group on the DNA that can then be removed by Apn1, Apn2, and Tpp1 (Vance and Wilson 2001). When Tdp1 is not available, a variety of structure-specific endonucleases, including Rad1, Slx4, Mre11, and Mus81, may be employed to remove the DNA ends covalently bound to the Top1 protein complex (Figure 6).
As these endonucleases have different substrate preferences, on the basis of biochemical evidence it suggests that a variety of DNA structures may arise from stalled replication forks caused by camptothecin. Rad1, Slx4, and Mus81 all act on branched DNA structures, but also have their unique substrate specificities. While Rad1 prefers the simple Y flap, Mus81 preferentially cuts the duplex flap and replication fork (de Laat et al. 1998; Kaliraman et al. 2001; Bastin-Shanower et al. 2003). We initially hypothesized that the involvement of Slx4 in Top1 removal would demonstrate the importance of the Slx1-Slx4 complex, as it has been shown that Slx4 stimulates Slx1 5′ flap nuclease activity (Fricke and Brill 2003). However, the resistance of the slx1 tdp1 double null mutant to camptothecin argues against this possibility (Figure 1b). It therefore seems likely that the observed stimulation of Slx1 5′ flap nuclease activity is only one function of Slx4 in vitro. In vivo, Slx4 may be responsible for a nuclease activity either by itself or with another protein. The finding that Slx4 has weak endonuclease activity on several sites of a simple Y substrate appears to support this (Fricke and Brill 2003). However, this activity is unlikely to be the mechanism for its in vivo role in Top1 removal as suggested by our result, because the cleavage sites by Slx4 alone in vitro are all distal to the branch point; i.e., they are on the overhanging single strand. Since Top1 is attached to the 3′ end of DNA, one would expect the cleavage sites proximal (5′) to the branching point on the duplex to be more effective in removing Top1, as seen with Rad1 and Mus81 (de Laat et al. 1998; Kaliraman et al. 2001; Bastin-Shanower et al. 2003). An exception to this scenario is that the presence of Top1 on the 3′ DNA ends causes significant melting of the duplex DNA either through physical displacement caused by the bulky Top1 protein complex or by a helicase or through a 5′-3′ exonuclease on the other strand.
In contrast, Mre11 has single-strand DNA (ssDNA) endonuclease and dsDNA and ssDNA 3′-5′ exonuclease activities (Usui et al. 1998). The mre11-H125N mutant lacks endonuclease activity but keeps weak 3′-5′ exonuclease activity (Moreau et al. 1999). The resulting mutant cannot process double-strand breaks (DSBs) in meiosis, where the DNA 5′ ends are covalently attached to Spo11, but can still process the “clean” DSBs generated by HO endonuclease (Moreau et al. 1999). This suggests that Mre11 endonuclease activity is essential to process the meiotic DSBs that have a protein complex attached at the 5′ end. In meiosis, the ssDNA substrate for Mre11 endonuclease activity is thought to be generated by an exonuclease activity or other proteins capable of unwinding duplex DNA, thereby allowing the endonuclease to remove the 5′ attached Spo11. Thus the 3′-5′ exonuclease activity of Mre11 is likely to be important in meiosis but it would not be effective in the case of Top1 attached to the 3′ DNA end. A 5′-3′ exonuclease is probably more appropriate to produce the ssDNA substrate for Mre11 in this case. Alternatively, an unwinding protein could generate the ssDNA substrate for Mre11. In fact, all the endonucleases implicated in resecting the Top1 attached DNA strand would be more efficient with such unwinding activity. Although its nature is not known, Srs2 is a reasonable candidate for this activity. The srs2 deletion mutant is sensitive to camptothecin (Table 1 and Vance and Wilson 2002), and there is a synergism of camptothecin sensitivity between the srs2 and tdp1 mutants (Vance and Wilson 2002).
The role of Sae2 in Top1 removal is most likely related to its interaction with Mre11, as in other cases where Sae2 functions have been examined (McKee and Kleckner 1997; Prinz et al. 1997; Moreau et al. 1999; Mullen et al. 2001), and our data showing that sae2 is epistatic to the exonuclease-defective mre11-H125N mutant (Figure 4b) suggests that Sae2 is a cofactor for Mre11 endonuclease activity. The sae2 and mre11-H125N mutants are similar to mus81 in that all are sensitive to camptothecin (Figures 3 and 4). They are also unable to process meiotic DSBs, and the mutants are sporulation deficient (McKee and Kleckner 1997; Prinz et al. 1997; Moreau et al. 1999; Mullen et al. 2001).
In summary, we have identified Slx4 and Mre11 as two endonucleases that protect against camptothecin toxicity, probably by removing Top1 from 3′ DNA ends. Thus, they constitute two more branches of repair by nucleases, in addition to Mus81 and Rad1. The function of Slx4 in repairing camptothecin damage is distinct from its known role as a partner and stimulator for the nuclease Slx1. The importance of the Mre11 endonuclease domain in removing Spo11 in meiosis and Top1 in mitosis suggests that Mre11, presumably with Rad50 and Xrs2, is active in both meiosis and mitosis and can repair both 5′ and 3′ DNA ends that are covalently attached to protein complexes.
We thank Lorraine S. Symington (Columbia University) for providing the mre11-H125N strain and Angela Chu (Stanford University) for providing the sae2, slx2, and slx4 deletion strains. This work was supported by National Institutes of Health grant P01 CA67166.
↵ 1 Present address: Department of Radiation Medicine, Second Military Medical University, 800 Xiangyin Rd., Shanghai, 200433, People's Republic of China.
Communicating editor: L. S. Symington
- Received March 15, 2004.
- Accepted March 4, 2005.
- Genetics Society of America