Requirement of the Mre11 Complex and Exonuclease 1 for Activation of the Mec1 Signaling Pathway

ABSTRACT The large protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate DNA damage checkpoint pathways. In budding yeast, ATM and ATR homologs are encoded by TEL1 and MEC1, respectively. The Mre11 complex consists of two highly related proteins, Mre11 and Rad50, and a third protein, Xrs2 in budding yeast or Nbs1 in mammals. The Mre11 complex controls the ATM/Tel1 signaling pathway in response to double-strand break (DSB) induction. We show here that the Mre11 complex functions together with exonuclease 1 (Exo1) in activation of the Mec1 signaling pathway after DNA damage and replication block. Mec1 controls the checkpoint responses following UV irradiation as well as DSB induction. Correspondingly, the Mre11 complex and Exo1 play an overlapping role in activation of DSB- and UV-induced checkpoints. The Mre11 complex and Exo1 collaborate in producing long single-stranded DNA (ssDNA) tails at DSB ends and promote Mec1 association with the DSBs. The Ddc1-Mec3-Rad17 complex associates with sites of DNA damage and modulates the Mec1 signaling pathway. However, Ddc1 association with DSBs does not require the function of the Mre11 complex and Exo1. Mec1 controls checkpoint responses to stalled DNA replication as well. Accordingly, the Mre11 complex and Exo1 contribute to activation of the replication checkpoint pathway. Our results provide a model in which the Mre11 complex and Exo1 cooperate in generating long ssDNA tracts and thereby facilitate Mec1 association with sites of DNA damage or replication block.

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The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage

Cell Division ◽

10.1186/s13008-021-00072-x ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Muhseena N. Katheeja ◽

Shankar Prasad Das ◽

Suparna Laha

Keyword(s):

Dna Damage ◽

Budding Yeast ◽

S Phase ◽

Wild Type ◽

Yeast Protein ◽

Repair Gene ◽

Replication Checkpoint ◽

Bud Emergence ◽

Checkpoint Pathway

Abstract Background The budding yeast protein Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This helicase is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage. Results G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with faster kinetics in chl1 mutants compared to wild-type cells. Also, more damage to DNA is observed in chl1 cells. The viability falls synergistically in rad24chl1 cells. The regulation of Chl1p on budding kinetics in G1 phase falls in line with Rad9p/Chk1p and shows a synergistic effect with Rad24p/Rad53p. rad9chl1 and chk1chl1 shows similar bud emergence as the single mutants chl1, rad9 and chk1. Whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24, rad53 and chl1. In presence of MMS induced damage, synergistic with Rad24p indicates Chl1p’s role as a checkpoint at G1/S acting parallel to damage checkpoint pathway. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further, we have also confirmed that the checkpoint defect functions in parallel to the damage checkpoint pathway of Rad24p. Conclusion Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint activity in presence of damage. This confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1p thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p for Rad53p activation when damaging agents perturb the DNA. Apart from checkpoint activation, it also regulates the budding kinetics as a repair gene.

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The budding yeast protein Chl1p is required for delaying progression through G1/S phase after DNA damage.

10.21203/rs.3.rs-151524/v1 ◽

2021 ◽

Author(s):

Katheeja Muhseena N. ◽

Shankar Prasad Das ◽

Suparna Laha

Keyword(s):

Dna Damage ◽

Budding Yeast ◽

S Phase ◽

Wild Type ◽

Yeast Protein ◽

Replication Checkpoint ◽

Bud Emergence ◽

Checkpoint Pathway ◽

M Phase

Abstract Background: The helicase Chl1p is a nuclear protein required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination, ageing and plays an instrumental role in chromatin remodeling. This budding yeast protein is known to preserve genome integrity and spindle length in S-phase. Here we show additional roles of Chl1p at G1/S phase of the cell cycle following DNA damage. Results: G1 arrested cells when exposed to DNA damage are more sensitive and show bud emergence with a faster kinetics in chl1 mutants compared to wild-type cells. This role of Chl1p in G1 phase is Rad9p dependent and independent of Rad24 and Rad53. rad9chl1 shows similar bud emergence as the single mutants chl1 and rad9 whereas rad24chl1 and rad53chl1 shows faster bud emergence compared to the single mutants rad24 , rad53 and chl1 . In case of damage induced by genotoxic agent like hydroxyurea, Chl1p acts as a checkpoint at G1/S. The faster movement of DNA content through G1/S phase and difference in phosphorylation profile of Rad53p in wild type and chl1 cells confirms the checkpoint defect in chl1 mutant cells. Further we have observed that the checkpoint defect is synergistic with the replication checkpoint Sgs1p and functions in prallel to the checkpoint pathway of Rad24p. Conclusion: Chl1p shows Rad53p independent bud emergence and Rad53p dependent checkpoint, confirms its requirement in two different pathways to maintain the G1/S arrest when cells are exposed to damaging agents. The bud emergence kinetics and DNA segregation were similar to wild type when given the same damage in nocodazole treated chl1 cells which establishes the absence of any role of Chl1p at the G2/M phase. The novelty of this paper lies in revealing the versatile role of Chl1p in checkpoints as well as repair towards regulating G1/S transition. Chl1 thus regulates the G1/S phase by affecting the G1 replication checkpoint pathway and shows an additive effect with Rad24p as well as Rad53p activation when damaging agents perturbs the DNA.

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Autorepression of Rfx1 Gene Expression: Functional Conservation from Yeast to Humans in Response to DNA Replication Arrest

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10665-10673.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10665-10673 ◽

Cited By ~ 40

Author(s):

Yoav Lubelsky ◽

Nina Reuven ◽

Yosef Shaul

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Common Mechanism ◽

Animal Cells ◽

Replication Checkpoint ◽

Functional Conservation ◽

Replication Arrest ◽

Checkpoint Pathway ◽

Genes Encoding ◽

Replication Block

ABSTRACT The yeast Saccharomyces cerevisiae Crt1 transcription repressor is an effector of the DNA damage and replication checkpoint pathway. Crt1 binds and represses genes encoding ribonucleotide reductase (RNR) and its own promoter, establishing a negative-feedback pathway. The role of Rfx1, the mammalian Crt1 homologue, remained uncertain. In this study we investigated the possibility that Rfx1 plays a similar function in animal cells. We show here that, like Crt1, Rfx1 binds and represses its own promoter. Furthermore, Rfx1 binding to its promoter is reduced upon induction of a DNA replication block by hydroxyurea, which led to a release of repression. Significantly, like Crt1, Rfx1 binds and represses the RNR-R2 gene. Upon blocking replication and UV treatment, expression of both Rfx1 and RNR-R2 is induced; however, unlike the results seen with the RNR-R2 gene, the derepression of the RFX1 gene is only partially blocked by inhibiting Chk1, the DNA checkpoint kinase. This report provides evidence for a common mechanism for Crt1 and Rfx1 expression and for the conservation of their mode of action in response to a DNA replication block. We suggest that Rfx1 plays a role in the DNA damage response by down-regulating a subset of genes whose expression is increased in response to replication blocking and UV-induced DNA damage.

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Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

Molecular and Cellular Biology ◽

10.1128/mcb.17.6.3037 ◽

1997 ◽

Vol 17 (6) ◽

pp. 3037-3046 ◽

Cited By ~ 59

Author(s):

D Pati ◽

C Keller ◽

M Groudine ◽

S E Plon

Keyword(s):

Dna Damage ◽

Replication Checkpoint ◽

Site Analysis ◽

G2 Checkpoint ◽

Fork Head ◽

Checkpoint Pathway ◽

Dna Binding Site ◽

Acid Domain ◽

G2 Delay ◽

Amino Acid Domain

A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast.

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DNA Damage-Dependent Nuclear Dynamics of the Mre11 Complex

Molecular and Cellular Biology ◽

10.1128/mcb.21.1.281-288.2001 ◽

2001 ◽

Vol 21 (1) ◽

pp. 281-288 ◽

Cited By ~ 257

Author(s):

Olga K. Mirzoeva ◽

John H. J. Petrini

Keyword(s):

Dna Damage ◽

Cellular Response ◽

Human Fibroblasts ◽

Strand Break ◽

Immunofluorescent Localization ◽

Nuclear Retention ◽

Atm Kinase ◽

Mre11 Complex ◽

Previous Demonstration

ABSTRACT The Mre11 complex has been implicated in diverse aspects of the cellular response to DNA damage. We used in situ fractionation of human fibroblasts to carry out cytologic analysis of Mre11 complex proteins in the double-strand break (DSB) response. In situ fractionation removes most nucleoplasmic protein, permitting immunofluorescent localization of proteins that become more avidly bound to nuclear structures after induction of DNA damage. We found that a fraction of the Mre11 complex was bound to promyelocyte leukemia protein bodies in undamaged cells. Within 10 min after gamma irradiation, nuclear retention of the Mre11 complex in small granular foci was observed and persisted until 2 h postirradiation. In light of the previous demonstration that the Mre11 complex associated with ionizing radiation (IR)-induced DSBs, we infer that the protein retained under these conditions was associated with DNA damage. We also observed increased retention of Rad51 following IR treatment, although IR induced Rad51 foci were distinct from Mre11 foci. The ATM kinase, which phosphorylates Nbs1 during activation of the S-phase checkpoint, was not required for the Mre11 complex to associate with DNA damage. These data suggest that the functions of the Mre11 complex in the DSB response are implicitly dependent upon its ability to detect DNA damage.

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Pie1, a Protein Interacting with Mec1, Controls Cell Growth and Checkpoint Responses in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.21.3.755-764.2001 ◽

2001 ◽

Vol 21 (3) ◽

pp. 755-764 ◽

Cited By ~ 71

Author(s):

Tatsushi Wakayama ◽

Tae Kondo ◽

Seiko Ando ◽

Kunihiro Matsumoto ◽

Katsunori Sugimoto

Keyword(s):

Dna Damage ◽

Cell Growth ◽

Kinase Activity ◽

Critical Role ◽

Kinase Domain ◽

Protein Kinase Activity ◽

Replication Checkpoint ◽

Family Protein ◽

Replication Block

ABSTRACT In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Δ mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Δ cells, as in mec1Δcells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of thepie1Δ mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Δ mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

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The complex matter of DNA double-strand break detection

Biochemical Society Transactions ◽

10.1042/bst0310040 ◽

2003 ◽

Vol 31 (1) ◽

pp. 40-44 ◽

Cited By ~ 26

Author(s):

J.M. Bradbury ◽

S.P. Jackson

Keyword(s):

Dna Damage ◽

Genome Stability ◽

Strand Break ◽

Damage Sensing ◽

Dna Double Strand Break ◽

Mre11 Complex ◽

Damage Response ◽

Radiation Induced ◽

Dna Damage Signalling ◽

Complex Matter

To maintain genomic stability, despite constant exposure to agents that damage DNA, eukaryotic cells have developed elaborate and highly conserved pathways of DNA damage sensing, signalling and repair. In this review, we concentrate mainly on what we know about DNA damage sensing with particular reference to Lcd1p, a yeast protein that functions early in DNA damage signalling, and MDC1 (mediator of DNA damage checkpoint 1), a recently identified human protein that may be involved in recruiting the MRE11 complex to radiation-induced nuclear foci. We describe a model for the DNA damage response in which factors are recruited sequentially to sites of DNA damage to form complexes that can amplify the original signal and propagate it to the multitude of response pathways necessary for genome stability.

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Modeling Cancer Genomic Data in Yeast Reveals Selection Against ATM Function During Tumorigenesis

10.1101/771253 ◽

2019 ◽

Author(s):

Marcel Hohl ◽

Aditya Mojumdar ◽

Sarem Hailemariam ◽

Vitaly Kuryavyi ◽

Fiorella Ghisays ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Tumor Suppression ◽

Chromosome Instability ◽

Genomic Data ◽

Major Axis ◽

Strand Break ◽

Cancer Center ◽

Mre11 Complex

AbstractThe DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene-induced carcinogenesis. The complex is integral to all modes of double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S.cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was defective while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.Author SummaryA complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

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A pathway of targeted autophagy is induced by DNA damage in budding yeast

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614364114 ◽

2017 ◽

Vol 114 (7) ◽

pp. E1158-E1167 ◽

Cited By ~ 28

Author(s):

Vinay V. Eapen ◽

David P. Waterman ◽

Amélie Bernard ◽

Nathan Schiffmann ◽

Enrich Sayas ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Budding Yeast ◽

Strand Break ◽

Central Component ◽

Checkpoint Proteins ◽

Checkpoint Kinases ◽

Damage Response ◽

Autophagy Pathway ◽

Yeast Mutants

Autophagy plays a central role in the DNA damage response (DDR) by controlling the levels of various DNA repair and checkpoint proteins; however, how the DDR communicates with the autophagy pathway remains unknown. Using budding yeast, we demonstrate that global genotoxic damage or even a single unrepaired double-strand break (DSB) initiates a previously undescribed and selective pathway of autophagy that we term genotoxin-induced targeted autophagy (GTA). GTA requires the action primarily of Mec1/ATR and Rad53/CHEK2 checkpoint kinases, in part via transcriptional up-regulation of central autophagy proteins. GTA is distinct from starvation-induced autophagy. GTA requires Atg11, a central component of the selective autophagy machinery, but is different from previously described autophagy pathways. By screening a collection of ∼6,000 yeast mutants, we identified genes that control GTA but do not significantly affect rapamycin-induced autophagy. Overall, our findings establish a pathway of autophagy specific to the DNA damage response.

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Chl12 (Ctf18) Forms a Novel Replication Factor C-Related Complex and Functions Redundantly with Rad24 in the DNA Replication Checkpoint Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.21.17.5838-5845.2001 ◽

2001 ◽

Vol 21 (17) ◽

pp. 5838-5845 ◽

Cited By ~ 80

Author(s):

Takahiro Naiki ◽

Tae Kondo ◽

Daisuke Nakada ◽

Kunihiro Matsumoto ◽

Katsunori Sugimoto

Keyword(s):

Dna Replication ◽

Dna Damage Checkpoint ◽

Replication Factor C ◽

Replication Checkpoint ◽

Replication Factor ◽

Checkpoint Pathway ◽

Dna Replication Checkpoint ◽

Replication Block ◽

Factor C ◽

Related Complex

ABSTRACT RAD24 has been identified as a gene essential for the DNA damage checkpoint in budding yeast. Rad24 is structurally related to subunits of the replication factor C (RFC) complex, and forms an RFC-related complex with Rfc2, Rfc3, Rfc4, and Rfc5. Therad24Δ mutation enhances the defect ofrfc5-1 in the DNA replication block checkpoint, implicating RAD24 in this checkpoint.CHL12 (also called CTF18) encodes a protein that is structurally related to the Rad24 and RFC proteins. We show here that although neither chl12Δ norrad24Δ single mutants are defective, chl12Δ rad24Δ double mutants become defective in the replication block checkpoint. We also show that Chl12 interacts physically with Rfc2, Rfc3, Rfc4, and Rfc5 and forms an RFC-related complex which is distinct from the RFC and RAD24 complexes. Our results suggest that Chl12 forms a novel RFC-related complex and functions redundantly with Rad24 in the DNA replication block checkpoint.

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