Peroxiredoxins are a family of antioxidant enzymes critically involved in cellular defense and signaling. Particularly, yeast peroxiredoxin Tsa1p is thought to play a role in the maintenance of genome integrity, but the underlying mechanism is not understood. In this study, we took a genetic approach to investigate the cause of genome instability in tsa1Δ cells. Strong genetic interactions of TSA1 with DNA damage checkpoint components DUN1, SML1, and CRT1 were found when mutant cells were analyzed for either sensitivity to DNA damage or rate of spontaneous base substitutions. An elevation in intracellular dNTP production was observed in tsa1Δ cells. This was associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. In addition, defects in the DNA damage checkpoint did not modulate intracellular level of reactive oxygen species, but suppressed the mutator phenotype of tsa1Δ cells. On the contrary, overexpression of RNR1 exacerbated this phenotype by increasing dNTP levels. Taken together, our findings uncover a new role of TSA1 in preventing the overproduction of dNTPs, which is a root cause of genome instability.
Peroxiredoxins belong to a family of thiol-specific peroxidases widely and abundantly expressed in most living organisms [1], [2]. Through one or more redox-sensitive cysteines, peroxiredoxins not only scavenge reactive oxygen species (ROS) including peroxides and peroxynitrite [3], [4], but also function as an ROS sensor to regulate cell signaling [5]–[11]. For many peroxiredoxins, another level of regulation can be achieved through oligomerization [1], [2], [12]. In addition to their roles in peroxide reduction, peroxiredoxins are also known to possess chaperone activity [12], [13]. Loss-of-function studies in mice implicated an essential role of peroxiredoxins in antioxidant defense and tumor suppression [14]. Particularly, peroxiredoxin 1-knockout mice not only suffered from severe anemia due to oxidative stress, but were also susceptible to several types of malignant tumors [15]. Consistent with this, genome-wide screening revealed that yeast peroxiredoxin TSA1 was a strong suppressor of gross chromosomal rearrangements and spontaneous mutations [16], [17]. In addition, a mutator phenotype was observed in yeast cells lacking one or more peroxiredoxins. The phenotype could be rescued by yeast peroxiredoxin Tsa1p or mammalian Prx1, but not by their active-site mutants defective for peroxidase activity [18], [19]. In further support of a role of TSA1 in the maintenance of genome stability, many genetic interaction partners of TSA1 identified through synthetic genetic array analysis were components of DNA repair machinery or DNA checkpoints [20], [21]. For example, TSA1 was found to interact genetically with REV1/REV3 and OGG1, which are critically involved in translesion synthesis (TLS) and the repair of oxidative DNA damage, respectively [22], [23]. However, the exact mechanism by which Tsa1p suppresses genome instability remains to be fully understood. Intracellular dNTP levels are one important determinant of cellular response to DNA damage [24]. For yeast cells to survive DNA damage, increased dNTP production would be allowed to facilitate replication, but with a trade-off of high spontaneous mutation rate [25]. In other words, abnormally high dNTP levels are causally associated with genome instability [24], [26]. We previously demonstrated that yeast Tsa1p is a house-keeping peroxiredoxin which sufficiently suppressed the mutator phenotype [18]. Although both an aberrantly high level of ROS and an imbalance in free radical contents, which is caused by compensational activation of other antioxidants such as Sod1p [27], could underlie the mutator phenotype of tsa1Δ cells, additional events subsequent to the disruption of TSA1 might also be influential in the induction of genome instability. In this study we asked whether perturbation of dNTP pools might contribute to the mutator phenotype observed in tsa1Δ cells. We then investigated the cause of dNTP pool expansion. Our findings suggested that constitutive activation of the DNA damage checkpoint and consequent overproduction of dNTPs are the root cause of genome instability in tsa1Δ cells.
Yeast peroxiredoxin TSA1 was found to be a strong suppressor of mutations and gross chromosomal rearrangements [16]–[18]. In addition, further deletion of another gene involved in DNA repair or DNA checkpoints caused synthetic growth defect or lethality in tsa1Δ cells [21], [22]. Bearing these findings in mind, here we sought to dissect the interaction of TSA1 with the DNA damage checkpoint and particularly the machinery of dNTP synthesis, in order to understand the role of TSA1 in the maintenance of genome stability. We first examined the sensitivity of tsa1Δ cells to various DNA damaging agents. tsa1Δ cells were sensitive to hydroxyurea (HU), 4-nitroquinoline 1-oxide (4NQO) and ultraviolet (UV) irradiation (Figure 1A, lanes 1 and 5; Figure 1B; Figure 1C and Figure 1D, lanes 1 and 2). Re-expression of TSA1 in tsa1Δ cells suppressed the sensitivity phenotype (Figure 1C and Figure 1D, lane 3). This suppression required the catalytic cysteine (Cys47) of Tsa1p, but not the C-terminal cysteine (Cys170), pointing to the importance of the antioxidant property of Tsa1p in the protection against DNA damage (Figure 1C, lanes 3–5). The sensitivity of tsa1Δ cells to DNA damage prompted us to investigate further the genetic interactions between TSA1 and components of the DNA damage checkpoint. In light of the finding that TSA1 genetically interacts with DNA damage checkpoint genes DUN1 and SML1 [22], we chose these two genes and their effector CRT1 for further analysis. Dun1p is a checkpoint kinase that phosphorylates and regulates ribonucleotide reductase (RNR) inhibitor Sml1p [28]. Dun1p also inhibits Crt1p, a transcriptional corepressor of RNR, through phosphorylation [29], [30]. Deletion of DUN1, SML1 or CRT1 in tsa1Δ cells exerted a significant impact on their sensitivity to HU, 4NQO and UV irradiation. Loss of DUN1 further sensitized tsa1Δ cells to H2O2, HU, 4NQO and UV (Figure 1A, lanes 1,2, 5 and 6). In support of the specificity of effect, this sensitization was reversed upon expression of TSA1 or DUN1 in tsa1Δ dun1Δ cells (Figure 1D, lanes 6–8). Conversely, loss of SML1 or CRT1 rescued the sensitivity phenotype of tsa1Δ cells to 4NQO and UV (Figure 1A, lanes 5,7 and 8). It is noteworthy that such reversion of sensitivity was not observed in cells treated with H2O2 (Figure 1A, lanes 5,7 and 8), suggesting that the effect might be specific to DNA damaging agents and was not caused directly by ROS. These observations supported the notion that TSA1 interacts specifically with the DNA damage checkpoint in a manner that is not mediated directly through ROS. Although the sensitivity pattern of the different mutant strains in the spot assay was highly reproducible, a more quantitative comparison of these strains is desired. Hence, survival curves of strains in the presence of 4NQO and UV were also obtained (Figure 1B). Dose-dependent killing of the strains by 4NQO and UV was observed. At all doses tested, the degrees of sensitivity of different strains to either 4NQO or UV were in the same order as shown in the spot assay. In particular, the survival curves indicated a further enhancement of the sensitivity phenotype in tsa1Δ dun1Δ versus tsa1Δ cells and a suppression of sensitivity in tsa1Δ sml1Δ cells (Figure 1B). Collectively, our results demonstrated that the survival of tsa1Δ cells under DNA damage was decreased upon deletion of DUN1, but enhanced when either SML1 or CRT1 was genetically disrupted. We also compared the phenotypes of tsa1Δ cells and cells lacking Sod1p, another key antioxidant enzyme [31]. In contrast to the genetic interactions observed in tsa1Δ cells, deletion of DUN1, SML1 or CRT1 in sod1Δ cells enhanced its sensitivity to HU and 4NQO (Figure 1E, lanes 6,7 and 8). Thus, TSA1 and SOD1 interact with DUN1, SML1 and CRT1 through different mechanisms. We next investigated whether compromising DNA damage checkpoint genes in tsa1Δ cells might also alter their mutator phenotype. In agreement with previous reports [16]–[18], tsa1Δ cells exhibited high rates of spontaneous mutations in both canavanine-resistant (CANR) and 5FC-resistant (5FCR) assays (Figure 2A, Figure 2B and Figure 2D, groups 1 and 5). On the other hand, deletion of DUN1 did not significantly affect spontaneous mutation rates (Figure 2A, Figure 2B and Figure 2D, groups 1 and 2), whereas loss of SML1 or CRT1 caused a mild increase in CANR mutation rates in WT cells (Figure 2A and Figure 2D, groups 1,3 and 4). However, the disruption of DUN1, SML1 and CRT1 in tsa1Δ cells modulated the mutator phenotype in opposite directions (Figure 2A and Figure 2B, columns 5–8). Whereas reduction of spontaneous mutation rates was observed in tsa1Δ dun1Δ cells (Figure 2A and Figure 2B, columns 5 and 6), deletion of SML1 or CRT1 in tsa1Δ cells significantly enhanced the mutator phenotype (Figure 2A and Figure 2B, columns 1,7 and 8). Complementation of the reduction of mutation rate in tsa1Δ dun1Δ cells by re-introduction of DUN1 or TSA1 further verified the specificity of effect (Figure 2C, columns 6 and 8). Thus, the mutation rates of tsa1Δ cells correlated with the activity of the DNA damage checkpoint. In all individual deletion mutants, tsa1Δ cells displayed the highest mutation rate (Figure 2). We postulated that this might be attributed either directly or indirectly to the elevation of intracellular ROS levels in these cells [4]. If that is the case, challenging the other DNA damage checkpoint mutants with ROS might have an impact on the mutator phenotype. To test this idea, we treated the cells with low-dose H2O2 and assessed the impact on CANR mutation rates. Interestingly, mutation rates increased in WT and dun1Δ cells to comparable levels (Figure 2D, groups 1 and 2). In contrast, a further increase in mutability was observed when SML1 or CRT1 was comprised (Figure 2D, groups 1,3 and 4). Although the mutation rates of sml1Δ and crt1Δ cells in the presence of H2O2 were still not as high as that of tsa1Δ cells in the absence of H2O2 (Figure 2D, groups 3–5), our results did suggest that ROS could differentially modulate the mutator phenotype of different mutants. We next investigated the mechanism that underlies the correlation of DNA damage checkpoint activity in tsa1Δ cells with drug sensitivity and mutator phenotype (Figure 1 and Figure 2). DUN1, SML1 and CRT1 are regulators of RNR, the rate-limiting enzyme in dNTP synthesis [32]–[34]. Considered together with the model that elevated dNTP levels are required for surviving DNA damage in yeast at the price of increasing mutation rates [24], we asked whether the mutator phenotype of tsa1Δ cells would be due to alteration in cellular dNTP production. Thus, we measured dNTP levels in our mutants. Surprisingly, tsa1Δ cells produced significantly more dNTPs than wild type (WT) cells (Figure 3A, groups 1 and 5). The magnitude of dNTP overproduction in tsa1Δ cells was comparable to that in sml1Δ cells (Figure 3A, groups 3 and 5), in which the removal of Sml1p activates RNR leading to the rise in dNTP levels [28]. To shed further light on the roles of dNTP production in the generation of mutator phenotype, we compared the dNTP levels in other mutant cells. As expected, dun1Δ cells produced less dNTPs than WT cells (Figure 3A, groups 1 and 2), since Dun1p is required for phosphorylation and subsequent removal of the RNR inhibitor Sml1p [28]. Loss of CRT1 was also found to increase cellular dNTP production (Figure 3A, groups 1 and 4), as Crt1p is a transcriptional corepressor of RNRs [29]. However, loss of DUN1 reduced cellular dNTP production in tsa1Δ cells (Figure 3A, groups 5 and 6), whereas deletion of SML1 or CRT1 in tsa1Δ cells further increased dNTP levels (Figure 3A, groups 5,7 and 8). Noteworthily, increased production of dNTPs in tsa1Δ cells could be fully complemented by Tsa1p, but not by its catalytic cysteine mutant C47S (Figure 3B). Thus, the antioxidant property of Tsa1p was likely required for preventing overproduction of dNTPs. We then asked whether the reduction of dNTP pools in tsa1Δ dun1Δ cells would be associated with a further drop in intracellular ROS levels in the absence of DUN1. Interestingly, tsa1Δ dun1Δ cells exhibited a higher level of intracellular ROS over WT, tsa1Δ or dun1Δ cells (Figure 3C, columns 1,2, 5 and 6), suggesting that the mutator phenotype in tsa1Δ and tsa1Δ dun1Δ cells correlates directly with dNTP production, but not generation of ROS. On the other hand, loss of SML1 or CRT1 did not alter the ROS levels in either WT or tsa1Δ cells (Figure 3C, columns 1,3, 4,5, 7 and 8). Thus, in addition to the accumulation of ROS, elevation of dNTP production might also contribute to genome instability in tsa1Δ cells. While deletion of DUN1, SML1 or CRT1 has an impact on dNTP production, they are multifunctional proteins that might also affect other biological processes [28]–[30]. To address this concern, we modulated the production of dNTP more directly by overexpressing RNR1 gene in tsa1Δ and sod1Δ cells. This overexpression has previously been shown to elevate intracellular dNTP levels substantially [24], [26]. Indeed, when we induced the expression of Rnr1-3MYCp in WT cells (Figure 4A), the spontaneous mutation rate was increased (Figure 4B, columns 1 and 2). Furthermore, overexpression of RNR1 also exacerbated the mutator phenotype in tsa1Δ and sod1Δ cells (Figure 4B, columns 3–6). This lent additional support to the importance of dNTP overproduction in the induction of genome instability. If the mutability of tsa1Δ cells is indeed caused by dNTP overproduction, the mutations generated would primarily be base substitutions rather than large deletion and gross chromosomal rearrangements [24]. With this in mind, we examined the types of mutations arisen in tsa1Δ and other mutants (Table 1). We noted that the majority of mutations found in tsa1Δ cells were base substitutions (83. 3%) and frameshifts (13. 3%). Large deletions were very rare in tsa1Δ cells (3. 3%) when compared with WT (13. 3%) cells. In addition, all of the mutations found in tsa1Δ sml1Δ cells with high dNTP levels (Figure 3) were base substitutions (90%) and frameshifts (10%), whereas more deletions (13. 3%) were detected in tsa1Δ dun1Δ cells (Table 1) with low dNTP concentrations (Figure 3). In keeping with previous findings [16], relatively more deletions (10%) were also observed in sod1Δ cells (Table 1). Generally, base substitutions were more prevalent in the strain when dNTP levels were high (Figure 3), whereas the incidences of deletions correlated negatively with dNTP concentrations. Therefore, the mutation spectra of tsa1Δ and other strains are consistent with the notion that elevation of dNTP levels is the underlying cause of genome instability in the absence of TSA1. Above we demonstrated the elevation of dNTP levels in tsa1Δ cells (Figure 3). In addition, our results also indicated the genetic interaction of TSA1 with DNA checkpoint genes (Figure 1). This led us to further investigate whether elevated production of dNTPs in the absence of TSA1 might be explained by the activation of the DNA damage checkpoint. As a first step, we assessed checkpoint activation by examining the steady-state levels of Rad53p, the yeast ortholog of human CHK2 kinase whose phosphorylation and activation are pivotally involved in the control of checkpoint response to DNA damage [35], [36]. Particularly, Rad53p is a master regulator of Dun1p, Sml1p and Crt1p [35]. In this analysis we included the sod1Δ control strain, in which the effectors of the Mec1p-dependent DNA damage checkpoint were previously shown to be downregulated [31]. Phosphorylated Rad53p species were more evident in tsa1Δ cells compared to WT and sod1Δ cells (Figure 5A, lanes 1,3 and 5; Figure 5B, lanes 1 and 2; Figure 6A, lanes 1 and 2; and Figure 7A, lanes 1 and 3). This difference became more pronounced in the presence of H2O2 (Figure 5A, lanes 2,4 and 6; Figure 5B, lanes 5 and 6; Figure 6A, lanes 3 and 4; and Figure 7A, lanes 5 and 7). The steady-state levels of phosphorylated Rad53p in DNA damage checkpoint mutants were also compared. Whereas deletion of DUN1 triggered phosphorylation of Rad53p (Figure 5C, lanes 1 and 2), an observable increase in phosphorylated Rad53p species was not found in sml1Δ or crt1Δ cells (Figure 5C, lanes 1,3 and 4). Notably, loss of DUN1 in tsa1Δ cells further enhanced the activation of Rad53p (Figure 5C, lanes 5 and 6), whereas tsa1Δ sml1Δ and tsa1Δ crt1Δ cells had similar levels of phosphorylated Rad53p compared to tsa1Δ cells (Figure 5C, lanes 5,7 and 8). In addition to Rad53p, we also checked for the status of Rad9p, a more upstream transducer in the DNA damage checkpoint pathway [37]. Rad9-13MYCp was found to be activated in tsa1Δ cells (Figure 6B, lanes 1,2, 4 and 5) and this activation could not be reversed by the C47S mutant of Tsa1p (Figure 6B, lanes 2,3, 5 and 6). As a marker for DNA double-strand breaks (DSBs) [38], the level of γH2A was also found to be elevated in tsa1Δ cells as compared to WT (Figure 5B, lanes 1 and 2; Figure 6A, lanes 1 and 2; and Figure 6B, lanes 1 and 2). This agrees with a recent report that tsa1Δ cells displayed an increased number of Rad52-YFP foci indicative of DNA damage [23]. The levels of γH2A in other DNA damage checkpoint mutant cells were also examined. Among dun1Δ, sml1Δ and crt1Δ cells, an elevation in γH2A level was only found in dun1Δ cells (Figure 5C, lane 2). In addition, disruption of DUN1, SML1 or CRT1 in tsa1Δ cells did not affect γH2A levels significantly (Figure 5C, lanes 5–8). Noteworthily, although phosphorylated Rad53p and γH2A were abundant in dun1Δ and tsa1Δ dun1Δ cells (Figure 5C, lanes 2 and 6), their mutation rates remained low (Figure 2A and Figure 2B, columns 2 and 6) plausibly due to the low levels of dNTPs (Figure 3A, groups 2 and 6). In other words, elevation of dNTP levels might be the direct cause of genome instability. Consistent with the activation of Rad53p and Rad9p, the levels of Rad53p target Sml1-3HAp were diminished in tsa1Δ cells in the presence (Figure 5B, lanes 5 and 6) and absence of H2O2 (Figure 5B, lanes 1 and 2). Importantly, all of the above changes in tsa1Δ cells could be fully complemented by TSA1 (Figure 5B, lanes 2 and 3), but not by its C47S mutant (Figure 5B, lanes 2 and 4). On the other hand, in agreement with previous findings [31], we did not observe a significant change of Sml1p level in sod1Δ cells (data not shown). RNR is an important downstream effector of the DNA damage checkpoint which mediates the production of dNTPs [33], [34]. Since the expression of RNR genes is transcriptionally activated in response to DNA damage [29], we used semi-quantitative RT-PCR to determine the relative levels of RNR1/2/3/4 transcripts in the presence and absence of HU. In this analysis we included an additional control termed HUG1, a target of Mec1p induced highly by DNA damage [39]. As shown in Figure 6C, RNR transcripts were induced to higher levels in tsa1Δ cells than in WT and sod1Δ cells. The induction of RNR1 and RNR3 was greatest in both untreated and HU-treated tsa1Δ cells. The level of HUG1 transcript was also elevated in tsa1Δ cells and this was more pronounced in the presence of HU (Figure 6C). In sharp contrast, sod1Δ cells treated with HU showed a lower magnitude of induction of RNR1, RNR3 and HUG1 mRNAs (Figure 6C). These results obtained from sod1Δ cells were generally consistent with previous findings [31]. Thus, the pattern of RNR induction in tsa1Δ cells was not ascribed to a general effect caused by the lack of any antioxidant enzyme, but was highly specific. Collectively, our results suggested that loss of TSA1 induces the activation of the DNA damage checkpoint leading to the induction of RNR and consequent overproduction of dNTPs. If activation of the DNA damage checkpoint in tsa1Δ cells is really important to the generation of genome instability caused by dNTP overproduction, genetic disruption of the checkpoint would be able to reverse the mutator phenotype of tsa1Δ cells. To test this hypothesis, we employed a RAD53 mutant termed rad53AA, in which both T354 and T358 in the activation loop of Rad53p had been replaced by alanine, thereby abrogating the autophosphorylation activity in response to DNA damage [40]. This defective rad53AA allele similar to rad53-11 is thought to be associated with reduced dNTP production due to high abundance of Sml1p [40], [41]. Thus, we set out to characterize the phenotypes of tsa1Δ cells carrying the rad53AA allele. As documented previously [40], TSA1 rad53AA cells exhibited lower levels of phosphorylated Rad53p and higher abundance of Sml1-3HAp than TSA1 RAD53 cells (Figure 7A, lanes 1,2, 5 and 6). In response to H2O2, γH2A was induced to higher levels in all mutant cells (Figure 7A, lanes 5–8 compared to lanes 1–4). Notably, both tsa1Δ RAD53 and tsa1Δ rad53AA cells showed similar basal levels of γH2A (Figure 7A, lanes 3 and 4). Although stronger Rad53p activation was observed in tsa1Δ rad53AA cells, a more pronounced Sml1-3HAp protein band was seen (Figure 7A, lane 4 compared to lane 3), suggestive of a defective DNA damage checkpoint. We next characterized the sensitivity of these mutants towards H2O2, UV and HU. TSA1 rad53AA cells were sensitive to HU (Figure 7B, lanes 1 and 2) as previously described [40]; while tsa1Δ RAD53 cells were sensitive to H2O2, UV and HU (Figure 7B, lanes 1 and 3) similar to tsa1Δ cells in BY4741 background (Figure 1, lanes 1 and 5). Resembling tsa1Δ dun1Δ cells in BY4741, tsa1Δ rad53AA cells in W303 background displayed further sensitivity to H2O2 and UV when compared to tsa1Δ RAD53 cells (Figure 7B, lanes 3 and 4). We then looked at the effect of a defective DNA damage checkpoint on genome instability in tsa1Δ cells. Intriguingly, tsa1Δ rad53AA cells displayed a significantly reduced (∼50%) rate of spontaneous 5FCR mutations over tsa1Δ RAD53 cells (Figure 7C). On the other hand, both tsa1Δ rad53AA and tsa1Δ RAD53 cells had high levels of intracellular ROS over WT cells as measured by DCF fluorescence (Figure 7D). These observations suggested that rad53AA mutation can suppress the mutator phenotype in tsa1Δ cells without affecting cellular redox environment. This generally agrees with the phenotypes of tsa1Δ dun1Δ cells (Figure 1 and Figure 2), lending further support to the concept that intracellular dNTP levels are an important determinant in the induction of genome instability in tsa1Δ cells.
Here, we provided the first evidence that loss of yeast peroxiredoxin TSA1 causes genome instability through constitutive activation of the DNA damage checkpoint leading to overproduction of intracellular dNTPs. There are two salient points in our work. First, we demonstrated the elevation of dNTP levels in tsa1Δ cells and its direct correlation with the mutator phenotype (Figure 2, Figure 3, Figure 4). Second, we demonstrated the activation of the DNA damage checkpoint in tsa1Δ cells in relation to elevated production of dNTPs (Figure 1, Figure 5, Figure 6, Figure 7). Our findings suggested a new model for the role of peroxiredoxins in the maintenance of genome integrity, which has implications in the understanding of human diseases including cancer. In agreement with our findings on the accumulation of γH2A and activation of the DNA damage checkpoint in tsa1Δ cells, several lines of evidence in the literature supported the role of Tsa1p and other peroxiredoxins in the protection of cells against DNA damage. First, tsa1Δ cells produce significantly more ROS [4], which cause DNA and protein damage [27], [42], [43]. Second, loss of TSA1 results in increased formation of Rad52-YFP foci, an indicator of DNA DSBs [23]. Third, tsa1Δ cells are highly sensitive to the functional state of DNA repair and checkpoints [22]. In particular, tsa1Δ is synthetically lethal with rad51Δ mutation, indicating that the viability of rad51Δ cells deficient in recombination repair requires TSA1 function [44]. Finally, human peroxiredoxins have been implicated in cellular defense against oxidative DNA lesions [45]. In this context, the activation of the DNA damage checkpoint in tsa1Δ cells demonstrated in our study highlights the pivotal roles of the checkpoint in cell survival and provides an explanation for the synthetic lethality seen in various double deletion mutants involving TSA1 and another DNA repair or checkpoint gene [21]. Deletion of TSA1 in yeast cells has previously been shown to result in both a mutator phenotype and an increase in gross chromosomal rearrangements [16], [22], [23]. Although the causes and origin of gross chromosomal rearrangements remain poorly understood, oxygen metabolism and ROS production are implicated in the prevalence of these rearrangements in tsa1Δ cells [23]. Noteworthily, base substitution, but not chromosomal rearrangement, was the predominant type of mutation found in our analysis of mutation rates (Table 1). Thus, the major type of genome instability analyzed in our study is an increased rate of point mutations, but not gross chromosomal rearrangements involving more complex alterations such as translocations, large deletions and amplifications. Our findings point to a role of dNTP levels in determining the mutation rate of tsa1Δ cells. Strong genetic interactions between TSA1 and four RNR regulators DUN1, SML1, CRT1 and RAD53 were observed in the context of sensitivity to DNA damage (Figure 1 and Figure 7), spontaneous mutability (Figure 2 and Figure 7) and dNTP production (Figure 3). Although the catalytic cysteine of Tsa1p is required for the suppression of mutator phenotype, the mutability of tsa1Δ cells correlated directly with dNTP concentrations (Figure 2, Figure 3, Figure 4), but not with high ROS levels (Figure 3 and Figure 7). One plausible explanation is that the loss of TSA1 might cause accumulation of both ROS [4] and DNA damage (Figure 5). This activates the DNA damage checkpoint through Rad53p, Rad9p and Sml1p (Figure 5, Figure 6, Figure 7) leading to transcriptional activation of RNR genes (Figure 6) and elevated production of dNTPs (Figure 3). Once at high dNTP levels, replicative and TLS polymerases by-pass DNA lesions more efficiently to promote survival, but only at the price of increasing mutation rates [24], [25]. This model implicates dNTP pool expansion as the major culprit in the induction of genome instability in tsa1Δ cells. Indeed, reducing dNTP levels without affecting ROS production was sufficient to reverse the mutator phenotype of tsa1Δ cells (Figure 3). In particular, tsa1Δ dun1Δ cells have high levels of ROS (Figure 3C), phosphorylated Rad53p (Figure 5C) and γH2A (Figure 5C). However, these cells showed a low mutation rate (Figure 2C) because the dNTP levels were also low (Figure 3A). On the contrary, increasing dNTP levels by overexpressing RNR1 aggravated the mutator phenotype (Figure 4). Furthermore, point mutations but not deletions were predominantly found in tsa1Δ cells (Table 1), implicating a role for dNTP overproduction in compromising genome stability. In further support of this model, compromise of TLS polymerases also suppressed CANR mutations in tsa1Δ cells [23]. We found that the levels of dNTPs in tsa1Δ cells were as high as those in sml1Δ cells (Figure 3A). This finding revealed an unexpected role of TSA1 in the maintenance of dNTP pools in eukaryotic cells. We further observed transcriptional activation of RNR genes in tsa1Δ cells (Figure 6), which could be mediated through the activation of Rad53p checkpoint. Although this might provide an explanation for the overproduction of dNTPs, exactly how Tsa1p is mechanistically involved in regulating RNR expression remains to be further investigated. Consistent with previous findings [26], elevation of intracellular dNTPs over a particular threshold level by overexpressing Rnr1p can sufficiently induce a mutator phenotype (Figure 4). Plausibly, the dNTP levels in tsa1Δ sml1Δ and tsa1Δ crt1Δ cells might have reached the threshold level causing a dramatically increased mutation rate (Figure 2 and Figure 3). When the elevation of dNTP levels have not reached the threshold as in the case of sml1Δ, crt1Δ and tsa1Δ cells, accumulation of intracellular ROS might serve to trigger or aggravate the mutator phenotype. In tsa1Δ cells, ROS levels were constantly high (Figure 3C) causing severe DNA damage (Figure 5). In contrast, ROS levels were low (Figure 3C) and DNA damage was not detected (Figure 5C) in sml1Δ or crt1Δ cells. This might explain the higher mutation rate in tsa1Δ cells versus sml1Δ or crt1Δ cells (Figure 2). Further exacerbation of the mutator phenotype of sml1Δ and crt1Δ cells by ROS such as H2O2 (Figure 2D) lent some credence to this model. We demonstrated the requirement of the catalytic cysteine for the ability of Tsa1p to modulate dNTP production (Figure 3). Through irreversible hyperoxidation, this residue can act as a redox sensor, which triggers the switch of peroxiredoxin from peroxidase to chaperone activity under stress [12], [13]. In this connection, it would be of great interest to understand whether and how the chaperone activity of Tsa1p might be involved in the regulation of dNTP production. Activation of Rad53p by upstream kinase Mec1p requires adaptor proteins Rad9p and Mrc1p [46], [47]. We noted that Rad53p phosphorylation was dramatically increased in tsa1Δ versus WT cells (Figure 6A and Figure 7A). In contrast, the increase in Rad9p phosphorylation in the absence of TSA1 was less pronounced (Figure 6B). Although additional experiments are required to investigate the cause of this difference between Rad53p and Rad9p, one possibility is that the deletion of TSA1 might exert a stronger effect on Mrc1p activity. Hypermutability or genome instability is a hallmark of cancer [48]. Mammalian Prx1 is a candidate tumor suppressor gene [15], [49]. Because peroxiredoxins are highly evolutionarily conserved proteins, an understanding of the mechanism by which yeast Tsa1p protects cell from genome instability might derive novel insight into the tumor suppressive role of Prx1 in mammalian cells. Our work demonstrates the importance of high dNTP levels in the mutability of tsa1Δ cells. Further analysis of dNTP concentrations of Prx1-null mouse cells will reveal whether increased production of dNTPs might be a general mechanism for the generation of genome instability in higher eukaryotes.
S. cerevisiae strains BY4741 [50] and W303-1a, and their isogenic strains (Table 2) were used. All knockout mutants were constructed by one-step gene deletion method [51]. Primers were listed in Table 3. Expression vector for DUN1 was derived from pRS415. Expression plasmids for TSA1 and its mutants have been described [18]. Plasmid pGal-RNR1 kindly provided by Dr. Stephen Elledge has also been described previously [52]. Rates of spontaneous forward mutations to confer CANR or 5FCR were measured as described [18], [53]. Spectra of CANR mutations were determined by DNA sequencing. Ten independent cultures were analyzed in each experiment. Cell extracts were prepared and dNTP levels were measured with Klenow enzyme and [3H] labeled dATP or dTTP (PerkinElmer) as described [54]. Standard curves were used to estimate the cellular dNTP levels. Three independent cultures were analyzed in each experiment. Intracellular ROS levels were measured by fluorimetry using DCF (Molecular Probes) as described [4], [18]. Total RNA was extracted by phenol/freeze RNA preparation method as described [55]. For RT-PCR, 3 µg of total RNA was used for cDNA synthesis. Semi-quantitative PCR was performed and optimized to ensure that the amplification was in the linear range. PCR primers were listed in Table 3. Western blot analysis was performed essentially as described [31]. Yeast cells were harvested by centrifugation, followed by trichloroacetic acid extraction with the help of glass beads.
Peroxiredoxins are a family of antioxidant enzymes highly conserved from yeast to human. Loss of peroxiredoxin in mice can lead to severe anemia and malignant tumors, but the underlying cause is not understood. One way to derive new knowledge of peroxiredoxins is through genetic analysis in yeast. We have shown that loss of peroxiredoxins in yeast is associated with an increase in mutation rates. Here, we demonstrate that this elevation of mutation rates in yeast cells lacking a peroxiredoxin is due to increased production of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA. Our findings suggest a new model in which compromised antioxidant defense causes accumulation of damaged DNA and activation of the DNA damage checkpoint. For yeast cells to survive DNA damage, dNTP production is increased to facilitate DNA replication, but at the price of high mutation rates. This new model could lead to a better understanding of human diseases including cancer.
biochemistry/replication and repair
genetics and genomics/gene function
Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels