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1.
NAR Cancer ; 5(3): zcad042, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37554969

ABSTRACT

Targeting BRCA1- and BRCA2-deficient tumors through synthetic lethality using poly(ADP-ribose) polymerase inhibitors (PARPi) has emerged as a successful strategy for cancer therapy. PARPi monotherapy has shown excellent efficacy and safety profiles in clinical practice but is limited by the need for tumor genome mutations in BRCA or other homologous recombination genes as well as the rapid emergence of resistance. In this study, we identified 2-chloro-N,N-diethylethanamine hydrochloride (CDEAH) as a small molecule that selectively kills PARP1- and xeroderma pigmentosum A-deficient cells. CDEAH is a monofunctional alkylating agent that preferentially alkylates guanine nucleobases, forming DNA adducts that can be removed from DNA by either a PARP1-dependent base excision repair or nucleotide excision repair. Treatment of PARP1-deficient cells leads to the formation of strand breaks, an accumulation of cells in S phase and activation of the DNA damage response. Furthermore, CDEAH selectively inhibits PARP1-deficient xenograft tumor growth compared to isogenic PARP1-proficient tumors. Collectively, we report the discovery of an alkylating agent inducing DNA damage that requires PARP1 activity for repair and acts synergistically with PARPi.

2.
Cells ; 11(24)2022 12 16.
Article in English | MEDLINE | ID: mdl-36552858

ABSTRACT

Thyroid hormone receptor-interacting protein 13 (TRIP13) participates in various regulatory steps related to the cell cycle, such as the mitotic spindle assembly checkpoint and meiotic recombination, possibly by interacting with members of the HORMA domain protein family. Recently, it was reported that TRIP13 could regulate the choice of the DNA repair pathway, i.e., homologous recombination (HR) or nonhomologous end-joining (NHEJ). However, TRIP13 is recruited to DNA damage sites within a few seconds after damage and may therefore have another function in DNA repair other than regulation of the pathway choice. Furthermore, the depletion of TRIP13 inhibited both HR and NHEJ, suggesting that TRIP13 plays other roles besides regulation of choice between HR and NHEJ. To explore the unidentified functions of TRIP13 in the DNA damage response, we investigated its genome-wide interaction partners in the context of DNA damage using quantitative proteomics with proximity labeling. We identified MRE11 as a novel interacting partner of TRIP13. TRIP13 controlled the recruitment of MDC1 to DNA damage sites by regulating the interaction between MDC1 and the MRN complex. Consistently, TRIP13 was involved in ATM signaling amplification. Our study provides new insight into the function of TRIP13 in immediate-early DNA damage sensing and ATM signaling activation.


Subject(s)
DNA-Binding Proteins , Nuclear Proteins , DNA-Binding Proteins/metabolism , MRE11 Homologue Protein/genetics , Nuclear Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Damage , DNA
3.
Nat Commun ; 13(1): 6732, 2022 11 08.
Article in English | MEDLINE | ID: mdl-36347866

ABSTRACT

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.


Subject(s)
Amino Acyl-tRNA Synthetases , Isoleucine-tRNA Ligase , Isoleucine-tRNA Ligase/chemistry , Amino Acyl-tRNA Synthetases/metabolism , Glutamate-tRNA Ligase/chemistry , RNA, Transfer/metabolism
4.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: mdl-35217600

ABSTRACT

An ideal cancer therapeutic strategy involves the selective killing of cancer cells without affecting the surrounding normal cells. However, researchers have failed to develop such methods for achieving selective cancer cell death because of shared features between cancerous and normal cells. In this study, we have developed a therapeutic strategy called the cancer-specific insertions-deletions (InDels) attacker (CINDELA) to selectively induce cancer cell death using the CRISPR-Cas system. CINDELA utilizes a previously unexplored idea of introducing CRISPR-mediated DNA double-strand breaks (DSBs) in a cancer-specific fashion to facilitate specific cell death. In particular, CINDELA targets multiple InDels with CRISPR-Cas9 to produce many DNA DSBs that result in cancer-specific cell death. As a proof of concept, we demonstrate here that CINDELA selectively kills human cancer cell lines, xenograft human tumors in mice, patient-derived glioblastoma, and lung patient-driven xenograft tumors without affecting healthy human cells or altering mouse growth.


Subject(s)
CRISPR-Cas Systems , INDEL Mutation , Neoplasms/genetics , Animals , Cell Death/genetics , DNA Breaks, Double-Stranded , Heterografts , Humans , Mice
5.
Cell Cycle ; 19(15): 1952-1968, 2020 08.
Article in English | MEDLINE | ID: mdl-32594826

ABSTRACT

Centrosomes are the primary microtubule-organizing centers that are important for mitotic spindle assembly. Centrosome amplification is commonly observed in human cancer cells and contributes to genomic instability. However, it is not clear how centrosome duplication is dysregulated in cancer cells. Here, we report that ATAD5, a replisome protein that unloads PCNA from chromatin as a replication factor C-like complex (RLC), plays an important role in regulating centrosome duplication. ATAD5 is present at the centrosome, specifically at the base of the mother and daughter centrioles that undergo duplication. UAF1, which interacts with ATAD5 and regulates PCNA deubiquitination as a complex with ubiquitin-specific protease 1, is also localized at the centrosome. Depletion of ATAD5 or UAF1 increases cells with over-duplicated centrosome whereas ATAD5 overexpression reduces such cells. Consistently, the proportion of cells showing the multipolar mode of chromosome segregation is increased among ATAD5-depleted cells. The localization and function of ATAD5 at the centrosomes do not require other RLC subunits. UAF1 interacts and co-localizes with ID1, a protein that increases centrosome amplification upon overexpression. ATAD5 depletion reduces interactions between UAF1 and ID1 and increases ID1 signal at the centrosome, providing a mechanistic framework for understanding the role of ATAD5 in centrosome duplication.


Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , Centrosome/metabolism , DNA-Binding Proteins/metabolism , Inhibitor of Differentiation Protein 1/metabolism , Nuclear Proteins/metabolism , Animals , Cell Line , Centrioles/metabolism , Chromosome Segregation , Humans , Mice , Protein Binding , Replication Protein C/metabolism , S Phase
6.
Nat Commun ; 10(1): 5718, 2019 12 16.
Article in English | MEDLINE | ID: mdl-31844045

ABSTRACT

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.


Subject(s)
ATPases Associated with Diverse Cellular Activities/metabolism , DNA Replication/genetics , DNA-Binding Proteins/metabolism , Genomic Instability/genetics , Proliferating Cell Nuclear Antigen/metabolism , Rad51 Recombinase/metabolism , ATPases Associated with Diverse Cellular Activities/genetics , Bromodeoxyuridine/metabolism , Cell Line, Tumor , DNA Breaks/drug effects , DNA Repair , DNA Replication/drug effects , DNA-Binding Proteins/genetics , Flow Cytometry , Fluorescence Resonance Energy Transfer , Gene Knockdown Techniques , Genomic Instability/drug effects , HEK293 Cells , Humans , Hydroxyurea/pharmacology , Protein Binding/drug effects , RNA, Small Interfering/metabolism , Single Molecule Imaging
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