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Genetics, Vol. 169, 65-75, January 2005, Copyright © 2005
doi:10.1534/genetics.104.030940
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,2
* St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia
Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824
Department of Medicine SVH, The University of Melbourne, Fitzroy, Victoria 3065, Australia
2 Corresponding author: St. Vincent's Institute of Medical Research, 9 Princes St., Fitzroy, Victoria 3065, Australia.
E-mail: jheierhorst{at}svi.edu.au
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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phenotypes, including synthetic HU hypersensitivity with dun1, demonstrating that Ccr4-Not mRNA deadenylase activity is required for DNA damage responses. However, ccr4 and caf1 DNA damage phenotypes and genetic interactions with checkpoint genes are not identical, and deletions of some Not components that are believed to predominantly function at the transcriptional level rather than mRNA turnover, e.g., not5, also lead to increased DNA damage sensitivity and synthetic HU hypersensitivity with dun1. Taken together, our data thus suggest that both transcriptional and post-transcriptional functions of the Ccr4-Not complex contribute to the DNA damage response affecting gene expression in a complex manner.
In budding yeast, Mec1 and its subunit Ddc2 are the most upstream checkpoint kinases and may be able to sense some damage directly (KONDO et al. 2001; MELO et al. 2001; ROUSE and JACKSON 2002; ZOU and ELLEDGE 2003). Mec1 is required for three alternate DNA damage-signaling pathways that are characterized by the mediator proteins Mrc1, Rad9, and Rad17. While the Mrc1 pathway is believed to be specific for damage associated with DNA replication, the other two pathways are activated in response to a variety of lesions throughout the cell cycle (reviewed in NYBERG et al. 2002; LONGHESE et al. 2003). The mediators link Mec1 to the downstream effector kinases Rad53 and Chk1, thus enabling their activation (SANCHEZ et al. 1999; ALCASABAS et al. 2001; SCHWARTZ et al. 2002; OSBORN and ELLEDGE 2003). Together with Mec1, these kinases phosphorylate the checkpoint targets that execute the DNA damage response.
In addition to Rad53 and Chk1, Saccharomyces cerevisiae has a third effector kinase, Dun1, that acts mostly downstream of Rad53 in the checkpoint cascade. Dun1 has important roles in cell cycle arrest at the G2/M phase (GARDNER et al. 1999), transcriptional induction of damage-inducible genes [such as those coding for ribonucleotide reductase (RNR) subunits; ZHOU and ELLEDGE 1993; GASCH et al. 2001], phosphorylation and inhibition of the RNR inhibitor Sml1 (ZHAO and ROTHSTEIN 2002), and regulation of repair pathways (BASHKIROV et al. 2000). However, dun1 cells have higher genome instability rates than rad53 mutants (MYUNG et al. 2001), indicating Rad53-independent functions of Dun1. Similar to Rad53 and its mammalian homolog CHK2, Dun1 contains an N-terminal forkhead-associated (FHA) domain, a phosphothreonine-binding module present in a large number of proteins (reviewed in DUROCHER and JACKSON 2002; HAMMET et al. 2003). We recently reported that the FHA domain of Dun1 interacts with the Pan2-Pan3 poly(A) nuclease (HAMMET et al. 2002), a complex required for mRNA poly(A) tail length control (BROWN and SACHS 1998). Dun1 and Pan2-Pan3 act together in regulating mRNA levels of the DNA repair gene RAD5, and dun1 pan2 and dun1 pan3 mutants are synthetically lethal in the presence of replication blocks (HAMMET et al. 2002).
Shortening of the poly(A) tail by 3'
5' exonucleases is the first step in mRNA turnover (reviewed in PARKER and SONG 2004). In addition to Pan2-Pan3, yeast contains another mRNA deadenylase, the Ccr4-Not complex, which is the major deadenylase responsible for initiating mRNA degradation (DAUGERON et al. 2001; TUCKER et al. 2001). The Ccr4-Not complex is conserved throughout evolution and in yeast consists of nine defined subunits: Ccr4, Caf1, Not1–Not5, Caf40, and Caf130 (reviewed in COLLART 2003; DENIS and CHEN 2003). Ccr4 is the deadenylase catalytic subunit and contains a 3'5' exonuclease domain at its C terminus (CHEN et al. 2002; TUCKER et al. 2002). Single amino acid substitutions of catalytic residues in the Ccr4 exonuclease domain abolish its mRNA deadenylase activity in vivo and mimic most ccr4 phenotypes, indicating that its major cellular function is in mRNA degradation (CHEN et al. 2002; TUCKER et al. 2002). Although Caf1 also contains a nuclease domain and shows nuclease activity in vitro (DAUGERON et al. 2001; THORE et al. 2003), in vivo studies do not support the notion that it is the active nuclease within the Ccr4-Not complex (VISWANATHAN et al. 2004). Nevertheless, Caf1 is absolutely required for exonuclease activity of Ccr4 in vivo, as caf1 mutants show the same deadenylation defects as ccr4 (TUCKER et al. 2001).
Besides its role in regulating mRNA stability, Ccr4-Not also functions in the initiation and elongation phases of RNA polymerase II-dependent transcription (reviewed in COLLART 2003; DENIS and CHEN 2003). Binding and phenotypic analyses indicate that the complex can be physically and functionally divided into two parts, Ccr4-Caf1 and the Not proteins (BAI et al. 1999; MAILLET et al. 2000). Not1 is the core component of the complex and binds Ccr4 and Caf1 via its N terminus, while the other Not subunits are bound via the Not1 C terminus (BAI et al. 1999; MAILLET et al. 2000). Although both functional parts of the Ccr4-Not complex contribute to transcriptional as well as post-transcriptional functions, Ccr4-Caf1 seems to be predominantly involved in mRNA deadenylation, whereas the Not proteins are believed to be primarily involved in transcription (BADARINARAYANA et al. 2000; DENIS et al. 2001; TUCKER et al. 2002; reviewed in COLLART 2003).
Our finding that the poly(A) nuclease Pan2-Pan3 interacts with the checkpoint kinase Dun1 and has a critical role in survival of replication blocks indicated that post-transcriptional mechanisms of regulation of gene expression could be targeted by the DNA damage response pathway in yeast, and we have speculated that this could also involve the Ccr4-Not complex (HAMMET et al. 2002). An involvement of the Ccr4-Not in responding to DNA damage is supported by the fact that mutants in several of its subunits, including the mRNA deadenylase catalytic subunit Ccr4, have been found to be sensitive to DNA-damaging agents in a number of whole-genome screens (BENNETT et al. 2001; HANWAY et al. 2002; PARSONS et al. 2004; WESTMORELAND et al. 2004). To extend these analyses, here we have further investigated the role of the Ccr4-Not complex and its mRNA deadenylase activity in cellular DNA damage responses.
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MATERIALS AND METHODS |
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For survival experiments over a 24-hr time course, overnight cultures were diluted to A600 = 0.2–0.3 and grown for 3 hr before HU was added to 0.1 M. Control samples were taken immediately before HU addition. Survival was followed over a period of 24 hr after addition of HU. Dilutions were plated on YPD plates and viable colonies were counted after 3–5 days depending on the growth rate of the strain. Survival was determined as a percentage of viable colonies relative to the control plate before HU addition. All incubations were done at 30°. For every strain, survival experiments were performed with at least two independent colonies.
Rad53 Western blots:
To analyze Rad53 phosphorylation, cells were HU treated as above, washed three times in YPD, and released into YPD for up to 3 hr as indicated in the figure legend. Protein extracts were prepared using 8 M urea cracking buffer and processed for Rad53 Western blotting as described by PIKE et al. (2001)(2003).
Northern blot analysis:
For analysis of gene expression, cells were grown in YPD overnight and diluted to A600 = 0.2–0.3. After 3 hr of growth, cells were treated with 0.1 M HU for another 3 hr. RNA isolation and Northern analysis were performed as described (HAMMET et al. 2000). Blots were probed with 32P-labeled RNR3 or ACT1 probes (PIKE et al. 2003). After exposure on PhosphorImager screens, the signals were quantified using Molecular Dynamics software. RNR3 levels were normalized to levels of ACT1, and levels of RNR3 message in HU-treated and untreated cells were calculated by comparing expression levels to basal levels of RNR3 expression in the wild type.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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and dun1 ccr4 strains were tested for their ability to grow on plates containing HU or MMS. As shown in Figure 1A, ccr4 mutants were sensitive to both agents. Similar to what we observed for Dun1 and Pan2-Pan3, the dun1 ccr4 double mutants showed a dramatic increase in sensitivity to HU compared to either single mutant, suggesting that CCR4 and DUN1 might act toward the same goal in the cellular response to replication blocks, but provide separate functions. In contrast, the dun1 ccr4 cells were not synthetically sensitive to MMS (Figure 1A), potentially placing CCR4 and DUN1 in the same genetic pathway that acts upon alkylating DNA damage.
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cells to respond to DNA damage. CAF1-deleted cells showed even higher sensitivity to HU than did ccr4 mutants and were also mildly sensitive to MMS (Figure 1A). Similar to dun1 ccr4, dun1 caf1 mutants showed synthetic sensitivity on HU plates. However, unlike dun1 ccr4, dun1 caf1 cells were also more sensitive to MMS than were the respective single mutants (Figure 1A).
Impaired colony formation on plates containing DNA-damaging agents could reflect growth defects or an inability of the mutants to survive upon damage to the genome. To distinguish between these possibilities, we assayed the dun1, ccr4, dun1 ccr4, caf1, and dun1 caf1 strains for their ability to survive exposure to HU over a 24-hr time course. As shown in Figure 1B, the wild type could proliferate even in the presence of 0.1 M HU. dun1, ccr4, and caf1 mutants did not multiply, but remained viable, as >65% of the cells were able to form colonies on YPD plates after being removed from HU. In contrast, for the dun1 ccr4 and dun1 caf1 double mutants only 1–5% of the cells were viable following 24 hr of exposure to HU. These data indicate that the observed synthetic sensitivity of the dun1 ccr4 and dun1 caf1 double mutants is due to HU-induced lethality. In other words, although Ccr4-Caf1 and Dun1 are required for cellular proliferation in the presence of replication blocks, only when Ccr4-Caf1 and Dun1-dependent functions are simultaneously deleted is exposure to HU actually lethal for the cells. Therefore, both Ccr4-Caf1 and Dun1 perform critical roles in survival after replication stress.
Complex genetic interactions of CCR4 and CAF1 with checkpoint mediators Rad9, Rad17, and Mrc1:
To further explore the role of Ccr4 and Caf1 in the DNA damage checkpoint response we performed similar double-mutant analyses of ccr4 and caf1 mutants with deletions of the checkpoint genes RAD9, RAD17, or MRC1. As shown in Figure 2A, deletion of RAD17 increased the sensitivity of ccr4 mutants on HU and also less severely on MMS, but had no effect on caf1 cells. In contrast, rad9 surprisingly led to moderately improved growth of ccr4 and caf1 strains on HU and, in the case of caf1, also on MMS (Figure 2A). Deletion of MRC1 in ccr4 or caf1 mutants mimicked the deletion of DUN1, in that both ccr4 mrc1 and caf1 mrc1 double mutants were more sensitive than the single mutants to HU, but only caf1 mrc1 also showed synthetic sensitivity to MMS (Figure 2B). The observed interactions with checkpoint proteins confirm that Ccr4 and Caf1 have a role in checkpoint pathways activated by DNA damage and replication blocks.
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under basal conditions (Figure 3A control plate). However, with respect to DNA damage sensitivity, the exonuclease mutant behavior was similar to that in the deletion strain: it was sensitive to HU and MMS and in combination with dun1 was synthetically sensitive to HU, but not to MMS (Figure 3A). We also performed quantitative survival assays in liquid 0.1 M HU cultures over a 24-hr time course (Figure 3B). In these assays, the ccr4-1 catalytic domain mutant had slightly better proliferation properties than the ccr4 strain, but the HU survival rates of the dun1 ccr4-1 double mutant were indistinguishable from those of dun1 ccr4 (Figure 3B), indicating that loss of mRNA exonuclease activity is responsible for the genetic interactions of ccr4 with dun1. Altogether, the identical synthetic HU hypersensitivity of dun1 ccr4-1 compared to dun1 ccr4 (Figure 3, A and B) and similar MMS hypersensitivity of ccr4-1 compared to ccr4 (Figure 3A) demonstrate that Ccr4 mRNA deadenylase activity plays a critical role in response to DNA damage.
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and caf1 differed in some of their DNA damage-sensitivity phenotypes. For example, caf1 is more HU hypersensitive than ccr4 (Figures 1 and 2) and compared to ccr4 has different synthetic interactions in response to MMS with dun1 and in response to low-dose HU treatment with mrc1 (Figure 2B). A possible explanation to resolve this paradox was that nondeadenylase functions of the Ccr4-Not complex could also contribute to the DNA damage response. The main role of the Not proteins is believed to be in the regulation of transcription through modulation of the function of the general transcription factor TFIID (reviewed in COLLART 2003), and the respective mutants show only very subtle defects in mRNA deadenylation (TUCKER et al. 2002).
Therefore, to investigate if the transcriptional functions of the complex could also be involved in the DNA damage response, we analyzed the DNA damage sensitivity of not1-2, not2, not3, not4 and not5 mutants. In plate assays, NOT2, NOT4, and NOT5 were required for normal cell growth in the presence of HU and, in the case of NOT5, also in the presence of MMS, while not1-2 and not3 mutants had normal growth properties under these conditions (Figure 4A). In 24-hr HU survival experiments, not2, not4, and not5 strains showed a phenotype similar to that of ccr4 and caf1 (Figure 1B) in that they did not proliferate but remained largely viable (Figure 4B). Similar to ccr4 and caf1 (Figure 1B), not5 exhibited a dramatic synthetic lethality phenotype in concert with dun1 in response to HU (Figure 4B). Together with some discrepant DNA damage-sensitivity phenotypes between ccr4 and caf1 (Figures 1 and 2), these data support the notion that deadenylase-independent functions of the Ccr4-Not complex contribute to the DNA damage response, most likely involving its established role in the regulation of transcription.
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(Figure 5). Rpb9 has been previously shown to be involved in transcription initiation and elongation and transcription-coupled DNA repair (TCR; HULL et al. 1995; HEMMING et al. 2000; LI and SMERDON 2002), and rpb9 mutants are hypersensitive to MMS and 
-irradiation (BENNETT et al. 2001; CHANG et al. 2002). As expected, rpb9 cells showed impaired growth properties on HU plates, but in contrast to ccr4 they did not exhibit a synthetic sensitivity phenotype with dun1 (in fact, dun1 partially suppressed the HU growth defect phenotype of rpb9, suggesting that Dun1-dependent checkpoint functions may be involved in delaying cell cycle progression in the presence of DNA lesions that are normally repaired by TCR)(Figure 5A). Liquid survival assays indicated that the growth defect of rpb9 cells was fully reversible even after 24 hr in 100 mM HU, and again rpb9 and dun1 were not synthetic lethal under these conditions (Figure 5B). Therefore, these results indicate that the genetic interactions between Ccr4-Not components and the Dun1 checkpoint kinase are specific for the functions of these proteins and not simply due to nonspecific effects of impaired transcription.
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, dun1 ccr4, caf1, and dun1 caf1 mutants to HU was that they cannot induce RNR genes upon replication stress. We hence tested if RNR3 was a target of Ccr4-Not. As expected dun1 cells were not able to significantly induce RNR3 after treatment with HU (Figure 6, A and B). However, ccr4 and caf1 were proficient in transcribing RNR3 upon replication stress, with ccr4 mutants showing even three to four fold higher levels of RNR3 expression in HU than the wild type (Figure 6, A and B). Moreover, deletion of CCR4, but not CAF1, restored the ability of dun1 cells to produce RNR3 mRNA upon replication stress at almost wild type levels (dun1 ccr4 +HU samples in Figure 6, A and B). Interestingly, although the ccr4-1 exonuclease domain mutant had DNA damage phenotypes similar to the complete deletion, it did not lead to increased HU-induced RNR3 expression by itself nor restoration of HU-induced RNR3 expression in dun1 cells (Figure 6, A and B).
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, dun1 ccr4, caf1, and dun1 caf1 mutants to HU is to look at suppression effects of deleting SML1. As shown in Figure 6C, sml1 was able to partially suppress ccr4 and caf1 growth defects on HU plates, but it did not suppress the synthetic HU hypersensitivity of dun1 ccr4 and dun1 caf1 strains.
Collectively, these data indicate that the compromised activity of RNR upon treatment with HU is not the reason for the hypersensitivity of ccr4, dun1 ccr4, caf1, and dun1 caf1 mutants to replication blocks.
Prolonged Rad53 activation in dun1 ccr4 mutants:
To test if the severe sensitivity of dun1 ccr4 mutants to HU could be a consequence of impaired checkpoint signaling, we analyzed Rad53 activation and inactivation kinetics in these strains. Rad53 is activated by phosphorylation that can be detected as slower mobility bands by Western blot analysis (PELLICIOLI et al. 1999), and in response to replication blocks the relative amount of shifted Rad53 correlates with the strength of the checkpoint signal (PIKE et al. 2004). In dun1 and dun1 ccr4 strains Rad53 was hyperphosphorylated compared to the wild type after 3 hr of 100 mM HU treatment, as well as after 16 hr HU treatment (Figure 7A). These data indicate that the increased HU lethality of dun1 ccr4 (Figure 1) is not the result of checkpoint failure. Interestingly, Rad53 activation was slightly reduced in ccr4 compared to the wild type (Figure 7A), presumably because higher RNR3 levels in this strain (Figure 6, A and B) lead to reduced replicative damage in response to the same dose of HU.
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and dun1 ccr4, it was possible that the decreased ability of the double mutant to form colonies after release from HU (Figure 1B) is the result of permanent cell cycle arrest due to irreversible checkpoint activation. To test this possibility, we monitored Rad53 inactivation in these strains after release from HU into normal medium (Figure 7B). In the wild type, the original Rad53 shift was approximately 50% reduced after 30 min and fully reversed after 1 hr. In contrast, Rad53 inactivation was delayed by 1 hr in the dun1 mutant and by another hour in the dun1 ccr4 double mutant (Figure 7B). These data indicate that dun1 ccr4 cells are significantly impaired in reversing checkpoint activation in the recovery from replication stress, but they are nevertheless able to fully turn off the checkpoint signal within 3 hr after HU release. As the 1-hr delay in Rad53 inactivation in dun1 ccr4 cells compared to dun1 (or 2 hr compared to wild type) would have only a minor effect on colony growth on HU-free plates after 3 days (Figure 1B), these results indicate that the increased HU-dependent lethality of the double mutant is not the result of a checkpoint recovery defect. |
DISCUSSION |
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and caf1 mutants behaved overall in a similar manner, we found a number of important differences in their DNA damage-sensitivity profiles and synthetic genetic interactions with checkpoint genes (Figures 1 and 2). These discrepancies indicated that mRNA deadenylase-independent functions of the complex may also contribute to the DNA damage response. As Ccr4-Caf1 is part of the Ccr4-Not complex that functions in transcriptional regulation of gene expression in addition to mRNA deadenylation, we analyzed the role of other Not complex components in the DNA damage response and found that not2, not4, and not5 also resulted in replication block hypersensitivity and, in the case of not5 as an example, in a synthetic phenotype with dun1 similar to ccr4 or caf1 (Figure 4). Altogether, the most straightforward explanation for our findings is that both the transcriptional and the post-transcriptional functions of the Ccr4-Not complex play important functions in the DNA damage response.
A remarkable feature of dun1 ccr4, dun1 caf1, dun1 not5 double mutants, as well as double mutants of dun1 with a single residue substitution in an exonuclease catalytic residue (ccr4-1), was that they not only failed to grow in the continuous presence of the replication blocking agent HU, but also were unable to recover from replicative damage in survival assays in liquid cultures (Figures 1B, 3B, and 4B). Although dun1 ccr4 mutants were considerably delayed in reversing HU-induced Rad53 phosphorylation as a molecular marker for checkpoint activation, they were able to inactivate Rad53 within 3 hr of release from HU (Figure 7B). This indicates that dun1 ccr4 cells were able to process the checkpoint-activating DNA lesions into "neutral" products that were no longer recognized by the checkpoint machinery, yet were unable to sustain normal cell viability. As dun1 cells already have dramatically increased genome instability rates under basal conditions (MYUNG et al. 2001), a plausible explanation for this phenotype could be that ccr4 exacerbates this property of dun1 in response to HU by redirecting the repair of replicative DNA damage into inappropriate pathways with extensive genome rearrangements and loss of genetic material and subsequent loss of viability.
Considering its role in gene expression, there are two main possible mechanisms by which defects in the Ccr4-Not complex could affect DNA repair in "presensitized" dun1 mutants. First, changes to the transcriptional process per se could affect DNA repair, as damage is known to be more efficiently repaired by the TCR pathway in transcribed genes than in nontranscribed genes. However, in contrast to ccr4, caf1, and not5, deletion of the nonessential RNA polymerase II subunit RBP9, which plays important roles in TCR and also gives rise to transcriptional defects, did not result in a synthetic HU hypersensitivity with dun1. In addition, because the ccr4-1 mutation most likely acts at the post-transcriptional level, a different mechanism seems more likely. This second mechanism could be that loss of some components of the Ccr4-Not complex in concert with dun1 leads to complex changes in cellular mRNA profiles that adversely affect the repair of replicative DNA lesions by shifting the equilibrium between opposing DNA repair pathways. A similar mechanism has been invoked in the case of the related dun1 pan2 or dun1 pan3 phenotypes, where the increased replication block sensitivity could be attributed to increased RAD5 expression (HAMMET et al. 2002), but here we did not find significantly elevated RAD5 mRNA levels in dun1 ccr4 strains (data not shown). Surprisingly, we found that HU-induced expression of RNR3 was actually increased in ccr4 mutants (Figure 6), which should reduce the number of stalled replication forks; yet, despite presumably fewer lesions, this mutation resulted in HU hypersensitivity. With two candidate "culprits" ruled out, it will be interesting to see in more comprehensive array experiments how the expression of DNA repair genes is affected in mutants of the Ccr4-Not complex.
As already mentioned, ccr4-not mutants have been identified as sensitive to DNA damage induced by UV, ionizing radiation (IR), HU, MMS, and other DNA-damaging agents in several large-scale studies (BENNETT et al. 2001; HANWAY et al. 2002; WESTMORELAND et al. 2004). Some differences between these reports and our data are probably due to strain differences, use of diploid vs. haploid strains, and different DNA-damaging agents and conditions. Diploid not3 cells were reported to be IR hypersensitive (WESTMORELAND et al. 2004), whereas haploids are not hypersensitive to HU or MMS (Figure 5). Also, we found that although the mutants in the NOT genes were sensitive to UV, ccr4 and caf1 were not (data not shown). WESTMORELAND et al. (2004) also showed that diploid ccr4 cells have an extended RAD9-dependent cell cycle arrest after IR. The Rad9 pathway is not usually activated by replication blocks, but can be indirectly activated if primary replicative damage is processed into lesions that are sensed by the cell cycle-wide DNA damage pathways (PIKE et al. 2004). Rad9 could therefore also contribute to Rad53 hyperphosphorylation and delayed recovery after HU release in dun1 and dun1 ccr4 cells (Figure 7), although deletion of RAD9 improved growth of ccr4 and caf1 cells in the continuous presence of HU and MMS only very modestly (Figure 2A). Our results extend the findings of the large-scale screens by establishing that the mRNA deadenylase function of Ccr4-Not is involved in the DNA damage response and by comprehensively analyzing the genetic interactions of the complex with the alternate checkpoint pathways. In addition, our data suggest that the role of Ccr4-Not complex in DNA damage responses also involves mRNA deadenylase-independent functions, mostly likely related to its functions in the regulation of transcription. Not4, a subunit that we found was required for HU tolerance, is a potential ubiquitin ligase (ALBERT et al. 2002) and therefore this activity could be another means for Ccr4-Not to influence the DNA damage response, in a manner dependent or independent of its roles in transcription and mRNA turnover.
It is established that pathways activated by DNA damage target gene expression, and it is becoming more and more evident that, in addition to the well-characterized regulation of transcription (ZHOU and ELLEDGE 1993; HUANG et al. 1998; GASCH et al. 2001), the DNA damage response also works by modulating post-transcriptional events in mRNA physiology. Emerging examples of post-transcriptional factors with links to the DNA damage response include the mammalian mRNA polyadenylation factor CstF (KLEIMAN and MANLEY 2001), the cytoplasmic Schizosaccharomyces pombe poly(A) polymerase Cid13 (SAITOH et al. 2002), the budding yeast poly(A) nuclease complex Pan2-Pan3 (HAMMET et al. 2002), and now the Ccr4-Caf1 mRNA deadenylase complex. A question that is still unanswered is how post-transcriptional events in the cytoplasm are targeted by the DNA damage response, since the major components of checkpoint signaling pathways reside in the nucleus. Ccr4-Not could play a role in physically connecting these two processes via its dual role in both nuclear transcription and cytoplasmic mRNA turnover. The relation between these two functions of Ccr4-Not it is not yet clear, but it has been proposed that the interaction with the transcription machinery enables Ccr4-Not to associate cotranscriptionally with mRNA, before its export to the cytoplasm (TUCKER et al. 2001). In such a way, the Ccr4-Not complex could enable the DNA damage signal to be transferred from the nucleus to the cytoplasm to act there in the post-transcriptional regulation of mRNA stability. This way, simultaneous targeting of nuclear transcriptional functions and cytoplasmic post-transcriptional functions of the Ccr4-Not complex by the checkpoint machinery could provide a means to facilitate a rapid switch in cellular mRNA profiles to adapt to the complex changes required for an efficient DNA damage response.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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