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1.
EMBO J ; 40(18): e107413, 2021 09 15.
Article in English | MEDLINE | ID: mdl-34346517

ABSTRACT

DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.


Subject(s)
DNA Repair , DNA Replication , Nuclear Proteins/metabolism , Signal Transduction , Small Ubiquitin-Related Modifier Proteins/metabolism , Transcription Factors/metabolism , Genomic Instability , Humans , Protein Binding , Protein Processing, Post-Translational , Sumoylation , Ubiquitin/metabolism , Ubiquitination
2.
J Biol Chem ; 295(25): 8350-8362, 2020 06 19.
Article in English | MEDLINE | ID: mdl-32350109

ABSTRACT

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.


Subject(s)
DNA Damage , DNA-Directed DNA Polymerase/metabolism , Amino Acid Motifs , Catalytic Domain , Cell Line, Tumor , DNA Repair , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Peptides/analysis , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Protein Inhibitors of Activated STAT/antagonists & inhibitors , Protein Inhibitors of Activated STAT/genetics , Protein Inhibitors of Activated STAT/metabolism , Proteomics , RNA Interference , RNA, Small Interfering/metabolism , Small Ubiquitin-Related Modifier Proteins/antagonists & inhibitors , Small Ubiquitin-Related Modifier Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
3.
EMBO J ; 38(21): e102361, 2019 10 04.
Article in English | MEDLINE | ID: mdl-31613024

ABSTRACT

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.


Subject(s)
Adenosine Triphosphatases/metabolism , Ataxin-3/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA-Binding Proteins/metabolism , Homeostasis , Nuclear Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Adenosine Triphosphatases/genetics , Ataxin-3/genetics , Cell Survival , Chromatin/genetics , DNA-Binding Proteins/genetics , Genomic Instability , HEK293 Cells , HeLa Cells , Humans , Nuclear Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Signal Transduction , Ubiquitin-Protein Ligases/genetics , Ubiquitination
4.
Mol Cell ; 75(3): 483-497.e9, 2019 08 08.
Article in English | MEDLINE | ID: mdl-31253574

ABSTRACT

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.


Subject(s)
BRCA1 Protein/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , Deubiquitinating Enzymes/genetics , Histone Chaperones/genetics , Neoplasms/genetics , Binding Sites/genetics , Carrier Proteins/genetics , Cell Nucleus/genetics , Cell Nucleus/immunology , Cytoplasm/genetics , Cytoplasm/immunology , DNA Breaks, Double-Stranded , DNA Repair/immunology , Deubiquitinating Enzymes/immunology , HeLa Cells , Humans , Immunity, Cellular/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Neoplasms/immunology , Nuclear Matrix-Associated Proteins/genetics , Protein Binding/genetics , Ubiquitin/genetics , Ubiquitin-Specific Proteases/genetics , Ubiquitination/genetics
5.
EMBO Rep ; 18(11): 1991-2003, 2017 11.
Article in English | MEDLINE | ID: mdl-29021206

ABSTRACT

Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA-binding factor in human cells. RADX binds ssDNA via an N-terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins.


Subject(s)
DNA Repair , DNA Replication , DNA, Single-Stranded/genetics , DNA-Binding Proteins/genetics , Replication Protein A/genetics , Binding Sites , Cell Line, Tumor , DNA Damage , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , HCT116 Cells , Humans , Models, Molecular , Osteoblasts/cytology , Osteoblasts/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Replication Protein A/chemistry , Replication Protein A/metabolism
6.
Carcinogenesis ; 35(10): 2214-23, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24903338

ABSTRACT

The INhibitor of Growth (ING) proteins are encoded as multiple isoforms in five ING genes (ING1 -5) and act as type II tumor suppressors. They are growth inhibitory when overexpressed and are frequently mislocalized or downregulated in several forms of cancer. ING1 and ING2 are stoichiometric members of histone deacetylase complexes, whereas ING3-5 are stoichiometric components of different histone acetyltransferase complexes. The INGs target these complexes to histone marks, thus acting as epigenetic regulators. ING proteins affect angiogenesis, apoptosis, DNA repair, metastasis and senescence, but how the proteins themselves are regulated is not yet clear. Here, we find a small ubiquitin-like modification (SUMOylation) of the ING1b protein and identify lysine 193 (K193) as the preferred ING1b SUMO acceptor site. We also show that PIAS4 is the E3 SUMO ligase responsible for ING1b SUMOylation on K193. Sequence alignment reveals that the SUMO consensus site on ING1b contains a phosphorylation-dependent SUMOylation motif (PDSM) and our data indicate that the SUMOylation on K193 is enhanced by the S199D phosphomimic mutant. Using an ING1b protein mutated at the major SUMOylation site (ING1b E195A), we further demonstrate that ING1b SUMOylation regulates the binding of ING1b to the ISG15 and DGCR8 promoters, consequently regulating ISG15 and DGCR8 transcription. These results suggest a role for ING1b SUMOylation in the regulation of gene transcription.


Subject(s)
Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Sumoylation , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Amino Acid Motifs , Cytokines/genetics , Gene Expression Regulation , Genes, Tumor Suppressor , HEK293 Cells , Humans , Lysine/metabolism , Poly-ADP-Ribose Binding Proteins , Promoter Regions, Genetic , Protein Inhibitors of Activated STAT/metabolism , RNA-Binding Proteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitins/genetics
7.
Cancer Lett ; 345(1): 1-16, 2014 Apr 01.
Article in English | MEDLINE | ID: mdl-24333729

ABSTRACT

ING genes (ING1-5) were identified has tumor suppressor genes. ING proteins are characterized as Type II TSGs since they are involved in the control of cell proliferation, apoptosis and senescence. They may also function as Type I TSGs since they are also involved in DNA replication and repair. Most studies have reported that they are frequently lost in human tumors and epigenetic mechanisms or misregulation of their transcription may be involved. Recently, studies have described that this loss may be caused by microRNA inhibition. Here, we summarize the current knowledge on ING functions, their involvement in tumor suppression and, in order to give a full assessment of the current knowledge, we review all the studies that have examined ING status in human cancers.


Subject(s)
Genes, Tumor Suppressor , Intracellular Signaling Peptides and Proteins/genetics , MicroRNAs/genetics , Neoplasms/genetics , Nuclear Proteins/genetics , Tumor Suppressor Proteins/genetics , Animals , Humans , Inhibitor of Growth Protein 1 , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Tumor Suppressor Proteins/metabolism
8.
Cell Mol Life Sci ; 70(20): 3753-72, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23412501

ABSTRACT

Inhibitor of Growth 1 (ING1) was identified and characterized as a "candidate" tumor suppressor gene in 1996. Subsequently, four more genes, also characterized as "candidate" tumor suppressor genes, were identified by homology search: ING2, ING3, ING4, and ING5. The ING proteins are characterized by a high homology in their C-terminal domain, which contains a Nuclear Localization Sequence and a Plant HomeoDomain (PHD), which has a high affinity to Histone 3 tri-methylated on lysine 4 (H3K4Me3). The ING proteins have been involved in the control of cell growth, senescence, apoptosis, chromatin remodeling, and DNA repair. Within the ING family, ING1 and ING2 form a subgroup since they are evolutionarily and functionally close. In yeast, only one gene, Pho23, is related to ING1 and ING2 and possesses also a PHD. Recently, the ING1 and ING2 tumor suppressor status has been fully established since several studies have described the loss of ING1 and ING2 protein expression in human tumors and both ING1 and ING2 knockout mice were reported to have spontaneously developed tumors, B cell lymphomas, and soft tissue sarcomas, respectively. In this review, we will describe for the first time what is known about the ING1 and ING2 genes, proteins, their regulations in both human and mice, and their status in human tumors. Furthermore, we explore the current knowledge about identified functions involving ING1 and ING2 in tumor suppression pathways especially in the control of cell cycle and in genome stability.


Subject(s)
Homeodomain Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , Apoptosis , Base Sequence , DNA Repair , DNA Replication , Gene Expression Regulation, Neoplastic , Genomic Instability , Homeodomain Proteins/genetics , Humans , Inhibitor of Growth Protein 1 , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Molecular Sequence Data , Nuclear Proteins/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Transcription, Genetic , Tumor Suppressor Proteins/genetics
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