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W2006150241 abstract "The yeast Dun1 kinase has complex checkpoint functions including DNA damage-dependent cell cycle arrest in G2/M, transcriptional induction of repair genes, and regulation of postreplicative DNA repair pathways. Here we report that the Dun1 forkhead-associated domain interacts with the Pan3 subunit of the poly(A)-nuclease complex and that dun1pan2and dun1pan3 double mutants are dramatically hypersensitive to replicational stress. This phenotype was independent of the function of Dun1 in regulating deoxyribonucleotide levels as it was also observed in strains lacking the ribonucleotide reductase inhibitor Sml1. dun1pan2 mutants initially arrested normally in response to replication blocks but died in the presence of persistent replication blocks with considerably delayed kinetics compared with mutants lacking the Rad53 kinase, indicating that the double mutation does not compromise the intra-S phase checkpoint. Interestingly, theRAD5 gene involved in error-free postreplication repair pathways was specifically up-regulated in dun1pan2 double mutants. Moreover, inducible overexpression of RAD5mimicked the double mutant phenotype by hypersensitizingdun1 mutants to replication blocks. The data indicate that Dun1 and Pan2-Pan3 cooperate to regulate the stoichiometry and thereby the activity of postreplication repair complexes, suggesting that posttranscriptional mechanisms complement the transcriptional response in the regulation of gene expression by checkpoint signaling pathways in Saccharomyces cerevisiae. The yeast Dun1 kinase has complex checkpoint functions including DNA damage-dependent cell cycle arrest in G2/M, transcriptional induction of repair genes, and regulation of postreplicative DNA repair pathways. Here we report that the Dun1 forkhead-associated domain interacts with the Pan3 subunit of the poly(A)-nuclease complex and that dun1pan2and dun1pan3 double mutants are dramatically hypersensitive to replicational stress. This phenotype was independent of the function of Dun1 in regulating deoxyribonucleotide levels as it was also observed in strains lacking the ribonucleotide reductase inhibitor Sml1. dun1pan2 mutants initially arrested normally in response to replication blocks but died in the presence of persistent replication blocks with considerably delayed kinetics compared with mutants lacking the Rad53 kinase, indicating that the double mutation does not compromise the intra-S phase checkpoint. Interestingly, theRAD5 gene involved in error-free postreplication repair pathways was specifically up-regulated in dun1pan2 double mutants. Moreover, inducible overexpression of RAD5mimicked the double mutant phenotype by hypersensitizingdun1 mutants to replication blocks. The data indicate that Dun1 and Pan2-Pan3 cooperate to regulate the stoichiometry and thereby the activity of postreplication repair complexes, suggesting that posttranscriptional mechanisms complement the transcriptional response in the regulation of gene expression by checkpoint signaling pathways in Saccharomyces cerevisiae. forkhead-associated poly(A)-nuclease hemagglutinin hydroxyurea methyl methanesulfonate ribonucleotide reductase yeast extract/peptone/glucose Eukaryotic cells contain highly conserved checkpoint signaling pathways that prevent genomic instability by regulating the cellular response to DNA damage and replication blocks. Checkpoints involve slowing or arresting the cell cycle until the damage is repaired, the transcriptional induction of repair enzymes, and the direct activation of repair processes (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar). The yeast Dun1 protein is a member of a family of protein kinases closely related to the human Chk2/HuCds1 kinase (2Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar,3Brown A.L. Lee C.H. Schwarz J.K. Mitiku N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (236) Google Scholar) that is mutated in a subset of patients suffering from the Li-Fraumeni multicancer syndrome (4Bell D.W. Varley J.M. Szydlo T.E. Kang D.H. Wahrer D.C. Shannon K.E. Lubratovich M. Verselis S.J. Isselbacher K.J. Fraumeni J.F. Birch J.M., Li, F.P. Garber J.E. Haber D.A. Science. 1999; 286: 2528-2531Crossref PubMed Scopus (748) Google Scholar). These kinases are characterized by the presence of at least one FHA1 domain, a protein-protein interaction module present in more than 200 different proteins (5Li J. Lee G. Van Doren S.R. Walker J.C. J. Cell Sci. 2000; 113: 4143-4149Crossref PubMed Google Scholar) that seems to specifically bind to phosphorylated amino acids (preferentially phosphothreonine) in target sequences (6Sun Z. Hsiao J. Fay D.S. Stern D.F. Science. 1998; 281: 272-274Crossref PubMed Scopus (336) Google Scholar, 7Liao H. Byeon I.J. Tsai M.D. J. Mol. Biol. 1999; 294: 1041-1049Crossref PubMed Scopus (97) Google Scholar, 8Liao H. Yuan C., Su, M.I. Yongkiettrakul S. Qin D., Li, H. Byeon I.J. Pei D. Tsai M.D. J. Mol. Biol. 2000; 304: 941-951Crossref PubMed Scopus (66) Google Scholar, 9Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar).dun1 mutant strains have a reduced replication block/DNA damage-dependent induction of repair genes (10Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (294) Google Scholar), a reduced cell cycle arrest function in the G2/M checkpoint (11Pati D. Keller C. Groudine M. Plon S.E. Mol. Cell. Biol. 1997; 17: 3037-3046Crossref PubMed Scopus (88) Google Scholar, 12Gardner R. Putnam C.W. Weinert T. EMBO J. 1999; 18: 3173-3185Crossref PubMed Scopus (144) Google Scholar), and increased rates of spontaneous chromosome rearrangements (13Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 14Fasullo M. Koudelik J. AhChing P. Giallanza P. Cera C. Genetics. 1999; 152: 909-919PubMed Google Scholar). The Dun1 kinase is activated through phosphorylation by checkpoint signals in a MEC1- andRAD53-dependent manner (15Navas T.A. Zhou Z. Elledge S.J. Cell. 1995; 80: 29-39Abstract Full Text PDF PubMed Scopus (370) Google Scholar), and its positive effect on transcription involves the phosphorylation and inactivation of the Crt1 transcriptional repressor (16Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). In contrast to the cell cycle arrest function of the S phase checkpoint that isRAD53-dependent and largelyDUN1-independent (12Gardner R. Putnam C.W. Weinert T. EMBO J. 1999; 18: 3173-3185Crossref PubMed Scopus (144) Google Scholar), DUN1 seems to play a much more crucial role than RAD53 in preventing gross chromosomal rearrangements (particularly de novo telomere additions to chromosome breakpoints) after spontaneous DNA replication errors during normal cell cycles, indicating that it has a specific function in regulating postreplicative DNA repair pathways (13Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar).In addition to the transcriptional control of gene expression, posttranscriptional mRNA modifications and mRNA decay pathways play crucial roles in the eukaryotic regulation of protein levels (17Tucker M. Parker R. Annu. Rev. Biochem. 2000; 69: 571-595Crossref PubMed Scopus (119) Google Scholar). A major posttranscriptional modification is the addition of the poly(A) tail at the 3′-end of pre-mRNAs that contributes to the regulation of protein translation (18Otero L.J. Ashe M.P. Sachs A.B. EMBO J. 1999; 18: 3153-3163Crossref PubMed Scopus (108) Google Scholar, 19Fortes P. Inada T. Preiss T. Hentze M.W. Mattaj I.W. Sachs A.B. Mol. Cell. 2000; 6: 191-196Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and mRNA stability (17Tucker M. Parker R. Annu. Rev. Biochem. 2000; 69: 571-595Crossref PubMed Scopus (119) Google Scholar). InSaccharomyces cerevisiae, mRNA degradation is usually initiated by the 3′ → 5′ exonucleolytic digest of the poly(A) tail by a cytoplasmic mRNA deadenylase containing the Ccr4 and Caf1 proteins (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). In addition to Ccr4-Caf1, poly(A) tail length distribution is also regulated by the poly(A)-nuclease (PAN) complex, which consists of the catalytic 135-kDa Pan2 subunit with sequence motifs characteristic of RNase D-like 3′ → 5′ exonucleases (21Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (202) Google Scholar) and the 72-kDa Pan3 subunit of unknown function. The primary function of PAN seems to be to “preset” poly(A) tails to message-specific lengths before or during the nucleocytoplasmic export of mRNAs (22Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Crossref PubMed Scopus (180) Google Scholar), but it also contributes to cytoplasmic mRNA turnover as an alternative or complementing pathway to Ccr4-Caf1 (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar).Here we report that Dun1 cooperates with PAN in the regulation ofRAD5 mRNA levels and cell survival in response to replicational stress. The data suggest that posttranscriptional mechanisms contribute to the regulation of gene expression by checkpoint signaling pathways.DISCUSSIONDUN1 plays a crucial role in preventing gross chromosomal rearrangements resulting from inappropriate repair pathways of spontaneous replicative DNA damage even in the absence of exogenous DNA-damaging or replication-blocking agents (13Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The data presented here indicate that the regulation of RAD5 mRNA levels by Dun1 in concert with PAN contributes to the function ofDUN1 in maintaining genome stability.dun1Δpan2Δ cells remain in S phase for considerable time (Fig. 3 B), activate Rad53 as a key player in the intra-S checkpoint normally (Fig. 3 C), and die after replication blocks with considerably delayed kinetics compared withrad53Δ cells (Fig. 2 B). At the same time, expression of the RAD5 gene, which plays a role in postreplicative DNA repair (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar), is specifically deregulated (Fig. 4) and sufficient to cause HU-dependent lethality in thedun1Δ background (Fig. 5), while the accumulation of cells with aberrant DNA contents in flow cytometry profiles (Fig.3 B) coincides temporally with the reduced viability in survival curves (Fig. 2 B). The most likely explanation for the dun1Δpan2Δ phenotype is, therefore, that these cells arrest normally in S phase while replicative DNA damage persists but that this damage is inappropriately repaired, which removes the checkpoint signal, allowing for subsequent cell division with loss of genetic material and concomitantly increased lethality.Interestingly, DUN1 has recently been indirectly linked to regulation of the RAD6 epistasis group of whichRAD5 is a member (38Datta A. Schmeits J.L. Amin N.S. Lau P.J. Myung K. Kolodner R.D. Mol. Cell. 2000; 6: 593-603Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Rad5 is a RING finger domain protein that mediates the interaction of the Lys-63-specific ubiquitin ligases Ubc13 and Mms2 with Rad18 and the Lys-48-specific ubiquitin ligase Rad6 (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). This heteromeric complex has been proposed to generate a signal that activates pathways for the repair of DNA double strand breaks by homologous recombination (i.e. “error-free”) instead of non-homologous end joining (i.e. “error-prone”) (34Xiao W. Chow B.L. Broomfield S. Hanna M. Genetics. 2000; 155: 1633-1641Crossref PubMed Google Scholar, 35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar, 36Brusky J. Zhu Y. Xiao W. Curr. Genet. 2000; 37: 168-174Crossref PubMed Scopus (127) Google Scholar). The same surfaces involved in the heteromeric interaction of Rad5 and Rad18 can also mediate homodimerization of the respective subunits and thereby dissociate the pentamer into Rad5-Ubc13-Mms2 and Rad6-Rad18 subcomplexes, which may generate a Rad6-dependent signal to activate an error-prone subpathway (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). According to this model, increased Rad5 protein levels in thedun1Δpan2Δ strain could indeed shift the equilibrium away from the heteromeric complex toward an error-prone postreplicative DNA repair pathway that causes the increased lethality (Fig. 6). This pathway seems to be particularly critical in the absence of DUN1 asRAD5 overexpression alone had only a subtle effect on replicational stress survival of wild type or pan2Δ strains (Fig. 5). This is conceivable given that DUN1 has several additional DNA damage repair functions, e.g.transcriptional induction of repair enzymes (16Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) and direct regulation of repair proteins such as Rad55 (39Bashkirov V.I. King J.S. Bashkirova E.V. Schmuckli-Maurer J. Heyer W.D. Mol. Cell. Biol. 2000; 20: 4393-4404Crossref PubMed Scopus (131) Google Scholar), which may renderdun1Δ mutants more sensitive to deleterious effects of either pan2/3 deletion or RAD5overexpression.As synthetic lethality is usually a property of genes whose products interact in a common biochemical pathway (31Hartman J.L.I. Garvik B. Hartwell L. Science. 2001; 291: 1001-1004Crossref PubMed Scopus (604) Google Scholar), the simplest interpretation of our data is that Dun1 and PAN interact at the posttranscriptional level to regulate RAD5 gene expression. While the two-hybrid data indicate that Dun1 interacts directly with Pan3, a key question remaining to be answered is whether PAN activity is regulated by Dun1 in response to replication blocks. Given the synthetic effect of the two genes, it is quite possible that Dun1 does not directly regulate PAN but another enzyme with a similar function,e.g. Ccr4-Caf1. In this scenario (Fig. 6), PAN would constitutively regulate RAD5 mRNA levels, whereas Ccr4-Caf1 would be strictly Dun1-dependent and therefore compensate for the loss of PAN as long as DUN1 is present. Ccr4-Caf1 has multiple functions as an mRNA deadenylase (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) and as part of the CCR4-NOT complex in control of transcriptional initiation and elongation (40Denis C.L. Chiang Y.-C. Cui Y. Chen J. Genetics. 2001; 158: 627-634Crossref PubMed Google Scholar). Tucker et al. (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) have suggested that one possible explanation for linking the cytoplasmic Ccr4-Caf1 deadenylase to the transcriptional machinery is that it needs to be cotranscriptionally loaded onto messenger ribonucleoprotein complexes. If this is so, it would transiently be in a complex with PAN during the initial trimming of the poly(A) tail. In this context, Pan3 binding by the Dun1 FHA domain could act as a transient scaffold to bring the kinase domain in contact with Ccr4-Caf1 to phosphorylate it and regulate its substrate specificity before export to the cytoplasm. Interestingly, Caf1 (also known as Pop2) has recently been shown to be regulated by phosphorylation in response to diauxic shifts by the Yak1 kinase, but it is unclear whether this affects its deadenylase function (41Moriya H. Shimizu-Yoshida Y. Omori A. Iwashita S. Katoh M. Sakai A. Genes Dev. 2001; 15: 1217-1228Crossref PubMed Scopus (118) Google Scholar). An important goal of future studies will be to elucidate the precise molecular mechanism by which Dun1 contributes to the regulation of RAD5 mRNA stability.Transcriptional control of gene expression (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar) and cotranscriptional regulation of mRNA 3′-end processing and polyadenylation reactions (42Kleimann F.E. Manley J.L. Cell. 2001; 104: 743-753Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) are established components of cell cycle checkpoints. Our results indicate that posttranscriptional mechanisms involving poly(A) tail length control are an additional checkpoint target in the regulation of gene expression. The Pan2-Pan3 and Ccr4-Caf1 poly(A)-exonucleases, the Dun1 kinase, and members of the RAD6 epistasis group have reasonably conserved orthologs in mammals (2Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar, 20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). Therefore, the checkpoint-dependent regulation of posttranscriptional control pathways may be not be restricted to yeast but may also contribute to the prevention of chromosome aberrations in mammalian cells. Eukaryotic cells contain highly conserved checkpoint signaling pathways that prevent genomic instability by regulating the cellular response to DNA damage and replication blocks. Checkpoints involve slowing or arresting the cell cycle until the damage is repaired, the transcriptional induction of repair enzymes, and the direct activation of repair processes (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar). The yeast Dun1 protein is a member of a family of protein kinases closely related to the human Chk2/HuCds1 kinase (2Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar,3Brown A.L. Lee C.H. Schwarz J.K. Mitiku N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (236) Google Scholar) that is mutated in a subset of patients suffering from the Li-Fraumeni multicancer syndrome (4Bell D.W. Varley J.M. Szydlo T.E. Kang D.H. Wahrer D.C. Shannon K.E. Lubratovich M. Verselis S.J. Isselbacher K.J. Fraumeni J.F. Birch J.M., Li, F.P. Garber J.E. Haber D.A. Science. 1999; 286: 2528-2531Crossref PubMed Scopus (748) Google Scholar). These kinases are characterized by the presence of at least one FHA1 domain, a protein-protein interaction module present in more than 200 different proteins (5Li J. Lee G. Van Doren S.R. Walker J.C. J. Cell Sci. 2000; 113: 4143-4149Crossref PubMed Google Scholar) that seems to specifically bind to phosphorylated amino acids (preferentially phosphothreonine) in target sequences (6Sun Z. Hsiao J. Fay D.S. Stern D.F. Science. 1998; 281: 272-274Crossref PubMed Scopus (336) Google Scholar, 7Liao H. Byeon I.J. Tsai M.D. J. Mol. Biol. 1999; 294: 1041-1049Crossref PubMed Scopus (97) Google Scholar, 8Liao H. Yuan C., Su, M.I. Yongkiettrakul S. Qin D., Li, H. Byeon I.J. Pei D. Tsai M.D. J. Mol. Biol. 2000; 304: 941-951Crossref PubMed Scopus (66) Google Scholar, 9Durocher D. Taylor I.A. Sarbassova D. Haire L.F. Westcott S.L. Jackson S.P. Smerdon S.J. Yaffe M.B. Mol. Cell. 2000; 6: 1169-1182Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar).dun1 mutant strains have a reduced replication block/DNA damage-dependent induction of repair genes (10Zhou Z. Elledge S.J. Cell. 1993; 75: 1119-1127Abstract Full Text PDF PubMed Scopus (294) Google Scholar), a reduced cell cycle arrest function in the G2/M checkpoint (11Pati D. Keller C. Groudine M. Plon S.E. Mol. Cell. Biol. 1997; 17: 3037-3046Crossref PubMed Scopus (88) Google Scholar, 12Gardner R. Putnam C.W. Weinert T. EMBO J. 1999; 18: 3173-3185Crossref PubMed Scopus (144) Google Scholar), and increased rates of spontaneous chromosome rearrangements (13Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 14Fasullo M. Koudelik J. AhChing P. Giallanza P. Cera C. Genetics. 1999; 152: 909-919PubMed Google Scholar). The Dun1 kinase is activated through phosphorylation by checkpoint signals in a MEC1- andRAD53-dependent manner (15Navas T.A. Zhou Z. Elledge S.J. Cell. 1995; 80: 29-39Abstract Full Text PDF PubMed Scopus (370) Google Scholar), and its positive effect on transcription involves the phosphorylation and inactivation of the Crt1 transcriptional repressor (16Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). In contrast to the cell cycle arrest function of the S phase checkpoint that isRAD53-dependent and largelyDUN1-independent (12Gardner R. Putnam C.W. Weinert T. EMBO J. 1999; 18: 3173-3185Crossref PubMed Scopus (144) Google Scholar), DUN1 seems to play a much more crucial role than RAD53 in preventing gross chromosomal rearrangements (particularly de novo telomere additions to chromosome breakpoints) after spontaneous DNA replication errors during normal cell cycles, indicating that it has a specific function in regulating postreplicative DNA repair pathways (13Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). In addition to the transcriptional control of gene expression, posttranscriptional mRNA modifications and mRNA decay pathways play crucial roles in the eukaryotic regulation of protein levels (17Tucker M. Parker R. Annu. Rev. Biochem. 2000; 69: 571-595Crossref PubMed Scopus (119) Google Scholar). A major posttranscriptional modification is the addition of the poly(A) tail at the 3′-end of pre-mRNAs that contributes to the regulation of protein translation (18Otero L.J. Ashe M.P. Sachs A.B. EMBO J. 1999; 18: 3153-3163Crossref PubMed Scopus (108) Google Scholar, 19Fortes P. Inada T. Preiss T. Hentze M.W. Mattaj I.W. Sachs A.B. Mol. Cell. 2000; 6: 191-196Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) and mRNA stability (17Tucker M. Parker R. Annu. Rev. Biochem. 2000; 69: 571-595Crossref PubMed Scopus (119) Google Scholar). InSaccharomyces cerevisiae, mRNA degradation is usually initiated by the 3′ → 5′ exonucleolytic digest of the poly(A) tail by a cytoplasmic mRNA deadenylase containing the Ccr4 and Caf1 proteins (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). In addition to Ccr4-Caf1, poly(A) tail length distribution is also regulated by the poly(A)-nuclease (PAN) complex, which consists of the catalytic 135-kDa Pan2 subunit with sequence motifs characteristic of RNase D-like 3′ → 5′ exonucleases (21Moser M.J. Holley W.R. Chatterjee A. Mian I.S. Nucleic Acids Res. 1997; 25: 5110-5118Crossref PubMed Scopus (202) Google Scholar) and the 72-kDa Pan3 subunit of unknown function. The primary function of PAN seems to be to “preset” poly(A) tails to message-specific lengths before or during the nucleocytoplasmic export of mRNAs (22Brown C.E. Sachs A.B. Mol. Cell. Biol. 1998; 18: 6548-6559Crossref PubMed Scopus (180) Google Scholar), but it also contributes to cytoplasmic mRNA turnover as an alternative or complementing pathway to Ccr4-Caf1 (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). Here we report that Dun1 cooperates with PAN in the regulation ofRAD5 mRNA levels and cell survival in response to replicational stress. The data suggest that posttranscriptional mechanisms contribute to the regulation of gene expression by checkpoint signaling pathways. DISCUSSIONDUN1 plays a crucial role in preventing gross chromosomal rearrangements resulting from inappropriate repair pathways of spontaneous replicative DNA damage even in the absence of exogenous DNA-damaging or replication-blocking agents (13Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The data presented here indicate that the regulation of RAD5 mRNA levels by Dun1 in concert with PAN contributes to the function ofDUN1 in maintaining genome stability.dun1Δpan2Δ cells remain in S phase for considerable time (Fig. 3 B), activate Rad53 as a key player in the intra-S checkpoint normally (Fig. 3 C), and die after replication blocks with considerably delayed kinetics compared withrad53Δ cells (Fig. 2 B). At the same time, expression of the RAD5 gene, which plays a role in postreplicative DNA repair (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar), is specifically deregulated (Fig. 4) and sufficient to cause HU-dependent lethality in thedun1Δ background (Fig. 5), while the accumulation of cells with aberrant DNA contents in flow cytometry profiles (Fig.3 B) coincides temporally with the reduced viability in survival curves (Fig. 2 B). The most likely explanation for the dun1Δpan2Δ phenotype is, therefore, that these cells arrest normally in S phase while replicative DNA damage persists but that this damage is inappropriately repaired, which removes the checkpoint signal, allowing for subsequent cell division with loss of genetic material and concomitantly increased lethality.Interestingly, DUN1 has recently been indirectly linked to regulation of the RAD6 epistasis group of whichRAD5 is a member (38Datta A. Schmeits J.L. Amin N.S. Lau P.J. Myung K. Kolodner R.D. Mol. Cell. 2000; 6: 593-603Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Rad5 is a RING finger domain protein that mediates the interaction of the Lys-63-specific ubiquitin ligases Ubc13 and Mms2 with Rad18 and the Lys-48-specific ubiquitin ligase Rad6 (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). This heteromeric complex has been proposed to generate a signal that activates pathways for the repair of DNA double strand breaks by homologous recombination (i.e. “error-free”) instead of non-homologous end joining (i.e. “error-prone”) (34Xiao W. Chow B.L. Broomfield S. Hanna M. Genetics. 2000; 155: 1633-1641Crossref PubMed Google Scholar, 35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar, 36Brusky J. Zhu Y. Xiao W. Curr. Genet. 2000; 37: 168-174Crossref PubMed Scopus (127) Google Scholar). The same surfaces involved in the heteromeric interaction of Rad5 and Rad18 can also mediate homodimerization of the respective subunits and thereby dissociate the pentamer into Rad5-Ubc13-Mms2 and Rad6-Rad18 subcomplexes, which may generate a Rad6-dependent signal to activate an error-prone subpathway (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). According to this model, increased Rad5 protein levels in thedun1Δpan2Δ strain could indeed shift the equilibrium away from the heteromeric complex toward an error-prone postreplicative DNA repair pathway that causes the increased lethality (Fig. 6). This pathway seems to be particularly critical in the absence of DUN1 asRAD5 overexpression alone had only a subtle effect on replicational stress survival of wild type or pan2Δ strains (Fig. 5). This is conceivable given that DUN1 has several additional DNA damage repair functions, e.g.transcriptional induction of repair enzymes (16Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) and direct regulation of repair proteins such as Rad55 (39Bashkirov V.I. King J.S. Bashkirova E.V. Schmuckli-Maurer J. Heyer W.D. Mol. Cell. Biol. 2000; 20: 4393-4404Crossref PubMed Scopus (131) Google Scholar), which may renderdun1Δ mutants more sensitive to deleterious effects of either pan2/3 deletion or RAD5overexpression.As synthetic lethality is usually a property of genes whose products interact in a common biochemical pathway (31Hartman J.L.I. Garvik B. Hartwell L. Science. 2001; 291: 1001-1004Crossref PubMed Scopus (604) Google Scholar), the simplest interpretation of our data is that Dun1 and PAN interact at the posttranscriptional level to regulate RAD5 gene expression. While the two-hybrid data indicate that Dun1 interacts directly with Pan3, a key question remaining to be answered is whether PAN activity is regulated by Dun1 in response to replication blocks. Given the synthetic effect of the two genes, it is quite possible that Dun1 does not directly regulate PAN but another enzyme with a similar function,e.g. Ccr4-Caf1. In this scenario (Fig. 6), PAN would constitutively regulate RAD5 mRNA levels, whereas Ccr4-Caf1 would be strictly Dun1-dependent and therefore compensate for the loss of PAN as long as DUN1 is present. Ccr4-Caf1 has multiple functions as an mRNA deadenylase (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) and as part of the CCR4-NOT complex in control of transcriptional initiation and elongation (40Denis C.L. Chiang Y.-C. Cui Y. Chen J. Genetics. 2001; 158: 627-634Crossref PubMed Google Scholar). Tucker et al. (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) have suggested that one possible explanation for linking the cytoplasmic Ccr4-Caf1 deadenylase to the transcriptional machinery is that it needs to be cotranscriptionally loaded onto messenger ribonucleoprotein complexes. If this is so, it would transiently be in a complex with PAN during the initial trimming of the poly(A) tail. In this context, Pan3 binding by the Dun1 FHA domain could act as a transient scaffold to bring the kinase domain in contact with Ccr4-Caf1 to phosphorylate it and regulate its substrate specificity before export to the cytoplasm. Interestingly, Caf1 (also known as Pop2) has recently been shown to be regulated by phosphorylation in response to diauxic shifts by the Yak1 kinase, but it is unclear whether this affects its deadenylase function (41Moriya H. Shimizu-Yoshida Y. Omori A. Iwashita S. Katoh M. Sakai A. Genes Dev. 2001; 15: 1217-1228Crossref PubMed Scopus (118) Google Scholar). An important goal of future studies will be to elucidate the precise molecular mechanism by which Dun1 contributes to the regulation of RAD5 mRNA stability.Transcriptional control of gene expression (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar) and cotranscriptional regulation of mRNA 3′-end processing and polyadenylation reactions (42Kleimann F.E. Manley J.L. Cell. 2001; 104: 743-753Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) are established components of cell cycle checkpoints. Our results indicate that posttranscriptional mechanisms involving poly(A) tail length control are an additional checkpoint target in the regulation of gene expression. The Pan2-Pan3 and Ccr4-Caf1 poly(A)-exonucleases, the Dun1 kinase, and members of the RAD6 epistasis group have reasonably conserved orthologs in mammals (2Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar, 20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). Therefore, the checkpoint-dependent regulation of posttranscriptional control pathways may be not be restricted to yeast but may also contribute to the prevention of chromosome aberrations in mammalian cells. DUN1 plays a crucial role in preventing gross chromosomal rearrangements resulting from inappropriate repair pathways of spontaneous replicative DNA damage even in the absence of exogenous DNA-damaging or replication-blocking agents (13Myung K. Datta A. Kolodner R.D. Cell. 2001; 104: 397-408Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). The data presented here indicate that the regulation of RAD5 mRNA levels by Dun1 in concert with PAN contributes to the function ofDUN1 in maintaining genome stability.dun1Δpan2Δ cells remain in S phase for considerable time (Fig. 3 B), activate Rad53 as a key player in the intra-S checkpoint normally (Fig. 3 C), and die after replication blocks with considerably delayed kinetics compared withrad53Δ cells (Fig. 2 B). At the same time, expression of the RAD5 gene, which plays a role in postreplicative DNA repair (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar), is specifically deregulated (Fig. 4) and sufficient to cause HU-dependent lethality in thedun1Δ background (Fig. 5), while the accumulation of cells with aberrant DNA contents in flow cytometry profiles (Fig.3 B) coincides temporally with the reduced viability in survival curves (Fig. 2 B). The most likely explanation for the dun1Δpan2Δ phenotype is, therefore, that these cells arrest normally in S phase while replicative DNA damage persists but that this damage is inappropriately repaired, which removes the checkpoint signal, allowing for subsequent cell division with loss of genetic material and concomitantly increased lethality. Interestingly, DUN1 has recently been indirectly linked to regulation of the RAD6 epistasis group of whichRAD5 is a member (38Datta A. Schmeits J.L. Amin N.S. Lau P.J. Myung K. Kolodner R.D. Mol. Cell. 2000; 6: 593-603Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Rad5 is a RING finger domain protein that mediates the interaction of the Lys-63-specific ubiquitin ligases Ubc13 and Mms2 with Rad18 and the Lys-48-specific ubiquitin ligase Rad6 (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). This heteromeric complex has been proposed to generate a signal that activates pathways for the repair of DNA double strand breaks by homologous recombination (i.e. “error-free”) instead of non-homologous end joining (i.e. “error-prone”) (34Xiao W. Chow B.L. Broomfield S. Hanna M. Genetics. 2000; 155: 1633-1641Crossref PubMed Google Scholar, 35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar, 36Brusky J. Zhu Y. Xiao W. Curr. Genet. 2000; 37: 168-174Crossref PubMed Scopus (127) Google Scholar). The same surfaces involved in the heteromeric interaction of Rad5 and Rad18 can also mediate homodimerization of the respective subunits and thereby dissociate the pentamer into Rad5-Ubc13-Mms2 and Rad6-Rad18 subcomplexes, which may generate a Rad6-dependent signal to activate an error-prone subpathway (35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). According to this model, increased Rad5 protein levels in thedun1Δpan2Δ strain could indeed shift the equilibrium away from the heteromeric complex toward an error-prone postreplicative DNA repair pathway that causes the increased lethality (Fig. 6). This pathway seems to be particularly critical in the absence of DUN1 asRAD5 overexpression alone had only a subtle effect on replicational stress survival of wild type or pan2Δ strains (Fig. 5). This is conceivable given that DUN1 has several additional DNA damage repair functions, e.g.transcriptional induction of repair enzymes (16Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar) and direct regulation of repair proteins such as Rad55 (39Bashkirov V.I. King J.S. Bashkirova E.V. Schmuckli-Maurer J. Heyer W.D. Mol. Cell. Biol. 2000; 20: 4393-4404Crossref PubMed Scopus (131) Google Scholar), which may renderdun1Δ mutants more sensitive to deleterious effects of either pan2/3 deletion or RAD5overexpression. As synthetic lethality is usually a property of genes whose products interact in a common biochemical pathway (31Hartman J.L.I. Garvik B. Hartwell L. Science. 2001; 291: 1001-1004Crossref PubMed Scopus (604) Google Scholar), the simplest interpretation of our data is that Dun1 and PAN interact at the posttranscriptional level to regulate RAD5 gene expression. While the two-hybrid data indicate that Dun1 interacts directly with Pan3, a key question remaining to be answered is whether PAN activity is regulated by Dun1 in response to replication blocks. Given the synthetic effect of the two genes, it is quite possible that Dun1 does not directly regulate PAN but another enzyme with a similar function,e.g. Ccr4-Caf1. In this scenario (Fig. 6), PAN would constitutively regulate RAD5 mRNA levels, whereas Ccr4-Caf1 would be strictly Dun1-dependent and therefore compensate for the loss of PAN as long as DUN1 is present. Ccr4-Caf1 has multiple functions as an mRNA deadenylase (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) and as part of the CCR4-NOT complex in control of transcriptional initiation and elongation (40Denis C.L. Chiang Y.-C. Cui Y. Chen J. Genetics. 2001; 158: 627-634Crossref PubMed Google Scholar). Tucker et al. (20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar) have suggested that one possible explanation for linking the cytoplasmic Ccr4-Caf1 deadenylase to the transcriptional machinery is that it needs to be cotranscriptionally loaded onto messenger ribonucleoprotein complexes. If this is so, it would transiently be in a complex with PAN during the initial trimming of the poly(A) tail. In this context, Pan3 binding by the Dun1 FHA domain could act as a transient scaffold to bring the kinase domain in contact with Ccr4-Caf1 to phosphorylate it and regulate its substrate specificity before export to the cytoplasm. Interestingly, Caf1 (also known as Pop2) has recently been shown to be regulated by phosphorylation in response to diauxic shifts by the Yak1 kinase, but it is unclear whether this affects its deadenylase function (41Moriya H. Shimizu-Yoshida Y. Omori A. Iwashita S. Katoh M. Sakai A. Genes Dev. 2001; 15: 1217-1228Crossref PubMed Scopus (118) Google Scholar). An important goal of future studies will be to elucidate the precise molecular mechanism by which Dun1 contributes to the regulation of RAD5 mRNA stability. Transcriptional control of gene expression (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar) and cotranscriptional regulation of mRNA 3′-end processing and polyadenylation reactions (42Kleimann F.E. Manley J.L. Cell. 2001; 104: 743-753Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) are established components of cell cycle checkpoints. Our results indicate that posttranscriptional mechanisms involving poly(A) tail length control are an additional checkpoint target in the regulation of gene expression. The Pan2-Pan3 and Ccr4-Caf1 poly(A)-exonucleases, the Dun1 kinase, and members of the RAD6 epistasis group have reasonably conserved orthologs in mammals (2Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar, 20Tucker M. Valencia-Sanchez M.A. Staples R.R. Chen J. Denis C.L. Parker R. Cell. 2001; 104: 377-386Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 35Ulrich H.D. Jentsch S. EMBO J. 2000; 19: 3388-3397Crossref PubMed Scopus (349) Google Scholar). Therefore, the checkpoint-dependent regulation of posttranscriptional control pathways may be not be restricted to yeast but may also contribute to the prevention of chromosome aberrations in mammalian cells. We thank Matthew O'Connell for help with tetrad dissections; Tony Tiganis for help with flow cytometry; Lindus Conlan for advice on two-hybrid screens; and Steve Dalton, Matthew O'Connell, Andy Poumbourios, and Tony Tiganis for critically reading the manuscript." @default.
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W2006150241 title "Posttranscriptional Regulation of the RAD5 DNA Repair Gene by the Dun1 Kinase and the Pan2-Pan3 Poly(A)-Nuclease Complex Contributes to Survival of Replication Blocks" @default.
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