The Yeast ULP2 (SMT4) Gene Encodes a Novel Protease Specific for the Ubiquitin-Like Smt3 Protein

ABSTRACT Yeast Smt3 and its vertebrate homolog SUMO-1 are ubiquitin-like proteins (Ubls) that are reversibly ligated to other proteins. LikeSMT3, SMT4 was first isolated as a high-copy-number suppressor of a defective centromere-binding protein. We show here that SMT4 encodes an Smt3-deconjugating enzyme, Ulp2. In cells lacking Ulp2, specific Smt3-protein conjugates accumulate, and the conjugate pattern is distinct from that observed in a ulp1ts strain, which is defective for a distantly related Smt3-specific protease, Ulp1. The ulp2Δ mutant exhibits a pleiotropic phenotype that includes temperature-sensitive growth, abnormal cell morphology, decreased plasmid and chromosome stability, and a severe sporulation defect. The mutant is also hypersensitive to DNA-damaging agents, hydroxyurea, and benomyl. Although cell cycle checkpoint arrest in response to DNA damage, replication inhibition, or spindle defects occurs with normal kinetics, recovery from arrest is impaired. Surprisingly, either introduction of aulp1ts mutation or overproduction of catalytically inactive Ulp1 can substantially overcome theulp2Δ defects. Inactivation of Ulp2 also suppresses several ulp1ts defects, and the double mutant accumulates far fewer Smt3-protein conjugates than either single mutant. Our data suggest the existence of a feedback mechanism that limits Smt3-protein ligation when Smt3 deconjugation by both Ulp1 and Ulp2 is compromised, allowing a partial recovery of cell function.

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Dual-utility NLS drives RNF169-dependent DNA damage responses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616602114 ◽

2017 ◽

Vol 114 (14) ◽

pp. E2872-E2881 ◽

Cited By ~ 29

Author(s):

Liwei An ◽

Yiyang Jiang ◽

Howin H. W. Ng ◽

Ellen P. S. Man ◽

Jie Chen ◽

...

Keyword(s):

Dna Damage ◽

Ring Finger ◽

Direct Interaction ◽

Cell Cycle Checkpoint ◽

High Fidelity ◽

Dna Double Strand Breaks ◽

Receptor Associated Protein ◽

Cycle Checkpoint ◽

Specific Protease ◽

Localization Signal

Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7–RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.

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Activation of p53-mediated cell cycle checkpoint in response to micronuclei formation

Journal of Cell Science ◽

10.1242/jcs.111.7.977 ◽

1998 ◽

Vol 111 (7) ◽

pp. 977-984

Author(s):

A.A. Sablina ◽

G.V. Ilyinskaya ◽

S.N. Rubtsova ◽

L.S. Agapova ◽

P.M. Chumakov ◽

...

Keyword(s):

Cell Cycle ◽

Chromosome Segregation ◽

Cell Activation ◽

P53 Gene ◽

Cell Cycle Checkpoint ◽

Abnormal Chromosome ◽

Brdu Incorporation ◽

Temperature Sensitive ◽

Cells With Micronuclei ◽

Cycle Checkpoint

Inactivation of p53 tumor-suppressor leads to genetic instability and, in particular, to accumulation of cells with abnormal numbers of chromosomes. In order to better define the role of p53 function in maintaining genome integrity we investigated the involvement of p53 in the control of proliferation of micronucleated cells resulting from abnormal chromosome segregation. Using cell lines expressing temperature-sensitive (ts) p53 or containing p53 genetic suppressor element (p53-GSE) we showed that inhibition of p53 function increases the frequency of cells with micronuclei. Immunofluorescence study revealed that in REF52 cell cultures with both spontaneous and colcemid-induced micronuclei the proportion of p53-positive cells is considerably higher among micronucleated variants as compared with their mononuclear counterparts. Analysis of 12(1)ConA cells expressing the beta-galactosidase reporter gene under the control of a p53-responsive promoter showed activation of p53-regulated transcription in the cells with micronuclei. Importantly, the percentage of cells manifesting specific p53 activity in colcemid-treated cultures increased with an augmentation of the number of micronuclei in the cell. Activation of p53 in micronucleated cells was accompanied by a decrease in their ability to enter S-phase as was determined by comparative analysis of 5-bromodeoxyuridine (5-BrdU) incorporation by the cells with micronuclei and their mononuclear counterparts. Inhibition of p53 function in the cells with tetracycline-regulated p53 gene expression, as well as in the cells expressing ts-p53 or p53-GSE, abolished cell cycle arrest in micronucleated cells. These results along with the data showing no increase in the frequency of chromosome breaks in REF52 cells after colcemid treatment suggest the existence of p53-mediated cell cycle checkpoint(s) preventing proliferation of micronucleated cells derived as a result of abnormal chromosome segregation during mitosis.

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Deletion of PIN4 Suppresses the Protein Transport Defects Caused by sec12-4 Mutation in Saccharomyces cerevisiae

Microbial Physiology ◽

10.1159/000509633 ◽

2020 ◽

Vol 30 (1-6) ◽

pp. 25-35

Author(s):

Akiko Murakami-Sekimata ◽

Masayuki Sekimata ◽

Natsumi Sato ◽

Yuto Hayasaka ◽

Akihiko Nakano

Keyword(s):

Protein Transport ◽

Cell Cycle Checkpoint ◽

Secretory Proteins ◽

Temperature Sensitive ◽

Nucleotide Exchange ◽

Casein Kinase I ◽

Vesicle Budding ◽

Nucleotide Exchange Factor ◽

Cycle Checkpoint ◽

Copii Vesicles

Newly synthesized secretory proteins are released into the lumen of the endoplasmic reticulum (ER). The secretory proteins are surrounded by coat protein complex II (COPII) vesicles, and transported from the ER and reach their destinations through the Golgi apparatus. Sec12p is a guanine nucleotide exchange factor for Sar1p, which initiates COPII vesicle budding from the ER. The activation of Sar1p by Sec12p and the subsequent COPII coat assembly have been well characterized, but the events that take place upstream of Sec12p remain unclear. In this study, we isolated the novel extragenic suppressor of sec12-4, PIN4/MDT1, a cell cycle checkpoint target. A yeast two-hybrid screening was used to identify Pin4/Mdt1p as a binding partner of the casein kinase I isoform Hrr25p, which we have previously identified as a modulator of Sec12p function. Deletion of PIN4 suppressed both defects of temperature-sensitive growth and the partial protein transport observed in sec12-4 mutants. The results of this study suggest that Pin4p provides novel aspects of Sec12p modulations.

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Identification of a Topoisomerase I Mutant, scsA1, as an Extragenic Suppressor of a Mutation in scaANBS1, the Apparent Homolog of Human Nibrin in Aspergillus nidulans

Genetics ◽

10.1093/genetics/164.3.935 ◽

2003 ◽

Vol 164 (3) ◽

pp. 935-945 ◽

Cited By ~ 1

Author(s):

Marcia R Z Kress Fagundes ◽

Larissa Fernandes ◽

Marcela Savoldi ◽

Steven D Harris ◽

Maria H S Goldman ◽

...

Keyword(s):

Dna Damage ◽

Aspergillus Nidulans ◽

Topoisomerase I ◽

Cell Cycle Checkpoint ◽

Dna Double Strand Breaks ◽

Dna Damaging Agents ◽

Cycle Checkpoint ◽

Increased Sensitivity ◽

Cellular Dna ◽

Extragenic Suppressors

Abstract The Mre11-Rad50-Nbs1 protein complex has emerged as a central player in the human cellular DNA damage response, and recent observations suggest that these proteins are at least partially responsible for the linking of DNA damage detection to DNA repair and cell cycle checkpoint functions. Mutations in scaANBS1, which encodes the apparent homolog of human nibrin in Aspergillus nidulans, inhibit growth in the presence of the antitopoisomerase I drug camptothecin. This article describes the selection and characterization of extragenic suppressors of the scaA1 mutation, with the aim of identifying other proteins that interfere with the pathway or complex in which the ScaA would normally be involved. Fifteen extragenic suppressors of the scaA1 mutation were isolated. The topoisomerase I gene can complement one of these suppressors. Synergistic interaction between the scaANBS1 and scsATOP1 genes in the presence of DNA-damaging agents was observed. Overexpression of topoisomerase I in the scaA1 mutant causes increased sensitivity to DNA-damaging agents. The scsATOP1 and the scaANBS1 gene products could functionally interact in pathways that either monitor or repair DNA double-strand breaks.

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Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring

Journal of Cell Science ◽

10.1242/jcs.109.1.131 ◽

1996 ◽

Vol 109 (1) ◽

pp. 131-142 ◽

Cited By ~ 43

Author(s):

F. Chang ◽

A. Woollard ◽

P. Nurse

Keyword(s):

Fission Yeast ◽

Ring Formation ◽

Cell Cycle Checkpoint ◽

Yeast Cells ◽

Temperature Sensitive ◽

Actin Ring ◽

Division Site ◽

Isolation And Characterization ◽

Cycle Checkpoint ◽

Actin Cables

Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a beta-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.

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RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

Molecular Biology of the Cell ◽

10.1091/mbc.6.10.1411 ◽

1995 ◽

Vol 6 (10) ◽

pp. 1411-1421 ◽

Cited By ~ 11

Author(s):

S Davey ◽

D Beach

Keyword(s):

Double Mutant ◽

Human Gene ◽

Cell Cycle Checkpoint ◽

Temperature Sensitive ◽

Uv Resistance ◽

Mitotic Entry ◽

Culture Cells ◽

Tissue Culture Cells ◽

Cycle Checkpoint ◽

Dose Dependent

We have identified a novel human gene by virtue of its ability to complement the rad1-1 checkpoint mutant of Schizosaccharomyces pombe. This gene, called RACH2, rescues the temperature-sensitive lethality of a rad1-1 wee1-50 double mutant of S. pombe. Expression of RACH2 in S. pombe rad1-1 strains partially restores UV resistance to the rad1-1 mutant strain. Expression of RACH2 in a rad1-1 cdc25-22 double mutant partially restores the dose-dependent delay in mitotic entry after irradiation that is lost in rad1-1 checkpoint-deficient mutants. Overexpression of RACH2 in human tissue culture cells induces apoptosis.

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