Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 6 de 6
Filter
Add more filters










Database
Language
Publication year range
1.
Cell ; 176(3): 505-519.e22, 2019 01 24.
Article in English | MEDLINE | ID: mdl-30612738

ABSTRACT

Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.


Subject(s)
Carrier Proteins/genetics , Nuclear Proteins/genetics , Carrier Proteins/metabolism , Chromatin/metabolism , DNA , DNA Breaks, Double-Stranded , DNA Damage/genetics , DNA End-Joining Repair , DNA-Binding Proteins/metabolism , Female , Genomic Instability , Germ-Line Mutation , Homologous Recombination , Humans , MRE11 Homologue Protein/genetics , MRE11 Homologue Protein/metabolism , Male , Neoplasms/genetics , Neoplasms/metabolism , Nuclear Proteins/metabolism , Primary Cell Culture , Recombinational DNA Repair
2.
Mol Cell ; 69(5): 866-878.e7, 2018 03 01.
Article in English | MEDLINE | ID: mdl-29499138

ABSTRACT

Double-strand breaks (DSBs) are critical DNA lesions that robustly activate the elaborate DNA damage response (DDR) network. We identified a critical player in DDR fine-tuning: the E3/E4 ubiquitin ligase UBE4A. UBE4A's recruitment to sites of DNA damage is dependent on primary E3 ligases in the DDR and promotes enhancement and sustainment of K48- and K63-linked ubiquitin chains at these sites. This step is required for timely recruitment of the RAP80 and BRCA1 proteins and proper organization of RAP80- and BRCA1-associated protein complexes at DSB sites. This pathway is essential for optimal end resection at DSBs, and its abrogation leads to upregulation of the highly mutagenic alternative end-joining repair at the expense of error-free homologous recombination repair. Our data uncover a critical regulatory level in the DSB response and underscore the importance of fine-tuning the complex DDR network for accurate and balanced execution of DSB repair.


Subject(s)
BRCA1 Protein/metabolism , Carrier Proteins/metabolism , DNA Breaks, Double-Stranded , Nuclear Proteins/metabolism , Recombinational DNA Repair/physiology , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/physiology , BRCA1 Protein/genetics , Carrier Proteins/genetics , DNA-Binding Proteins , HeLa Cells , Histone Chaperones , Humans , Nuclear Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitins/genetics , Ubiquitins/metabolism
3.
EMBO Rep ; 12(7): 713-9, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21637298

ABSTRACT

The cellular response to double-strand breaks (DSBs) in DNA is a complex signalling network, mobilized by the nuclear protein kinase ataxia-telangiectasia mutated (ATM), which phosphorylates many factors in the various branches of this network. A main question is how ATM regulates DSB repair. Here, we identify the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) as an ATM target. PNKP phosphorylates 5'-OH and dephosphorylates 3'-phosphate DNA ends that are formed at DSB termini caused by DNA-damaging agents, thereby regenerating legitimate ends for further processing. We establish that the ATM phosphorylation targets on human PNKP-Ser 114 and Ser 126-are crucial for cellular survival following DSB induction and for effective DSB repair, being essential for damage-induced enhancement of the activity of PNKP and its proper accumulation at the sites of DNA damage. These findings show a direct functional link between ATM and the DSB-repair machinery.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair Enzymes/metabolism , DNA Repair/physiology , DNA-Binding Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Cytotoxins/pharmacology , DNA Breaks, Double-Stranded/drug effects , DNA Repair Enzymes/genetics , HEK293 Cells , Humans , Mice , Phosphorylation/drug effects , Zinostatin/pharmacology
4.
Cell Mol Life Sci ; 67(12): 2025-38, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20204449

ABSTRACT

Mutation of tubulin chaperone E (TBCE) underlies hypoparathyroidism, retardation, and dysmorphism (HRD) syndrome with defective microtubule (MT) cytoskeleton. TBCE/yeast Pac2 comprises CAP-Gly, LRR (leucine-rich region), and UbL (ubiquitin-like) domains. TBCE folds alpha-tubulin and promotes alpha/beta dimerization. We show that Pac2 functions in MT dynamics: the CAP-Gly domain binds alpha-tubulin and MTs, and functions in suppression of benomyl sensitivity of pac2Delta mutants. Pac2 binds proteasomes: the LRR binds Rpn1, and the UbL binds Rpn10; the latter interaction mediates Pac2 turnover. The UbL also binds the Skp1-Cdc53-F-box (SCF) ubiquitin ligase complex; these competing interactions for the UbL may impact on MT dynamics. pac2Delta mutants are sensitive to misfolded protein stress. This is suppressed by ectopic PAC2 with both the CAP-Gly and UbL domains being essential. We propose a novel role for Pac2 in the misfolded protein stress response based on its ability to interact with both the MT cytoskeleton and the proteasomes.


Subject(s)
Hypoparathyroidism/genetics , Microtubules/metabolism , Molecular Chaperones , Proteasome Endopeptidase Complex/metabolism , Tubulin/metabolism , Amino Acid Sequence , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cytoskeleton/genetics , Cytoskeleton/metabolism , Dimerization , F-Box Proteins/genetics , F-Box Proteins/metabolism , F-Box-WD Repeat-Containing Protein 7 , Humans , Hypoparathyroidism/metabolism , Intellectual Disability/genetics , Intellectual Disability/metabolism , Intracellular Signaling Peptides and Proteins , Microtubules/genetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Chaperones/physiology , Mutation , Proteasome Endopeptidase Complex/genetics , Proteins/genetics , Proteins/metabolism , Syndrome , Tubulin/genetics , Ubiquitin/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
5.
Yeast ; 25(10): 733-43, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18949821

ABSTRACT

The yeast F-box protein Ufo1 recruits proteins for ubiquitylation by the SCF ubiquitin ligase complex preparing them for proteasomal degradation. Ufo1 has a role in maintenance of genome stability; its substrates include Ho endonuclease and Rad30 polymerase of error-prone DNA repair. Ufo1 is an unusual F-box protein, as it has three ubiquitin interacting motifs (UIMs). Deletion of the genomic UIMs is lethal; ectopic expression of UFO1 Delta UIMs extends protein half-life and arrests the cell cycle. A whole-genome study employing a TAP tag fused to the C-terminal UIMs did not identify Ufo1-interacting proteins. Here we therefore used stabilized N-terminally tagged Ufo1 Delta UIM as a strategy to identify Ufo1-interacting proteins by mass spectroscopy. We identified proteins that function in transcription, and an indirect interaction with Hsp70 molecular chaperones via the Skp1 adaptor; we also show that Ufo1 interacts with the 19S regulatory particle of the proteasome. Thus, our data augment the current network of known Ufo1 interacting proteins. We show directly that the UIMs are crucial for Ufo1 ubiquitylation in vivo, indicating that they facilitate turnover of SCF Ufo1 complexes. This allows recycling of the core subunits of the SCF complex and cell cycle progression.


Subject(s)
F-Box Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Adenosine Triphosphatases/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , HSP70 Heat-Shock Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Ubiquitin/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism
6.
Curr Genet ; 54(5): 271-81, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18807043

ABSTRACT

Exportin-5, an evolutionarily conserved nuclear export factor of the beta-karyopherin family, exports phosphorylated proteins and small noncoding RNAs. Msn5, the yeast ortholog, exports primarily phosphorylated cargoes including Ho endonuclease and a number of transcription factors and regulatory proteins. The Msn5-mediated nuclear export of Ho is dependent on phosphorylation of Thr225 by kinases of the DNA damage response pathway. Although Msn5 has been the object of many studies, no NES sequence capable of binding the exportin and/or of leading to Msn5-dependent export of a heterologous protein has been identified. Here we report identification of a 13-residue Ho sequence that interacts with Msn5 in vitro and directs Msn5-dependent nuclear export of GFP in vivo. A single point mutation in this 13-mer Ho NES abrogates both interaction with Msn5 and nuclear export of Ho and of GFP. However, this mutation, or of T225A, both of which abrogate nuclear export of Ho, does not interfere with its interaction with Msn5 implying that the exportin makes multiple contacts with its cargo. This can explain the lack of a conserved NES in Msn5 cargoes. Our results identify essential criteria for Msn5-mediated nuclear export of Ho: phosphorylation on HoT225, and interaction with the 13-mer Ho NES sequence.


Subject(s)
Cell Nucleus/enzymology , Deoxyribonucleases, Type II Site-Specific/metabolism , Karyopherins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Active Transport, Cell Nucleus , Amino Acid Sequence , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Conserved Sequence , Deoxyribonucleases, Type II Site-Specific/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Mutation , Phosphorylation , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...