scholarly journals UBE2T, the Fanconi Anemia Core Complex, and FANCD2 Are Recruited Independently to Chromatin: a Basis for the Regulation of FANCD2 Monoubiquitination

2007 ◽  
Vol 27 (24) ◽  
pp. 8421-8430 ◽  
Author(s):  
Arno Alpi ◽  
Frederic Langevin ◽  
Georgina Mosedale ◽  
Yuichi J. Machida ◽  
Anindya Dutta ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Fanconi Anemia ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Multiprotein Complex ◽  
Core Complex ◽  
Nuclear Core ◽  
Ubiquitin Conjugating Enzyme ◽  
Fancd2 Monoubiquitination

ABSTRACT The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.

DNA Damage-Induced Foci of E2 Ubiquitin-Conjugating Enzyme are Detectable upon Co-transfection with an Interacting E3 Ubiquitin Ligase

2015 ◽  
Vol 54 (2) ◽  
pp. 147-157 ◽  
Author(s):  
Degui Wang ◽  
Yingxia Tian ◽  
Dong Wei ◽  
Yuhong Jing ◽  
Haitao Niu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 723-723
Author(s):  
Alexandra Sobeck ◽  
Stacie Stone ◽  
Bendert deGraaf ◽  
Vincenzo Costanzo ◽  
Johan deWinter ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
S Phase ◽  
Dependent Manner ◽  
Core Complex ◽  
Damage Response ◽  
Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

scholarly journals Noncanonical E2 Variant-Independent Function of UBC13 in Promoting Checkpoint Protein Assembly

2008 ◽  
Vol 28 (19) ◽  
pp. 6104-6112 ◽  
Author(s):  
Michael S. Y. Huen ◽  
Jun Huang ◽  
Jingsong Yuan ◽  
Masahiro Yamamoto ◽  
Shizuo Akira ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Protein Assembly ◽  
Ring Domain ◽  
Dna Breaks ◽  
Focus Formation ◽  
Checkpoint Protein ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Conjugating Enzyme

ABSTRACT The E2 ubiquitin-conjugating enzyme UBC13 plays pivotal roles in diverse biological processes. Recent studies have elucidated that UBC13, in concert with the E3 ubiquitin ligase RNF8, propagates the DNA damage signal via a ubiquitylation-dependent signaling pathway. However, mechanistically how UBC13 mediates its role in promoting checkpoint protein assembly and its genetic requirement for E2 variants remain elusive. Here we provide evidence to support the idea that the E3 ubiquitin ligase complex RNF8-UBC13 functions independently of E2 variants and is sufficient in facilitating ubiquitin conjugations and accumulation of DNA damage mediator 53BP1 at DNA breaks. The RNF8 RING domain serves as the molecular platform to anchor UBC13 at the damaged chromatin, where localized ubiquitylation events allow sustained accumulation of checkpoint proteins. Intriguingly, we found that only a group of RING domains derived from E3 ubiquitin ligases, which have been shown to interact with UBC13, enabled UBC13-mediated FK2 and 53BP1 focus formation at DNA breaks. We propose that the RNF8 RING domain selects and loads a subset of UBC13 molecules, distinct from those that exist as heterodimers, onto sites of double-strand breaks, which facilitates the amplification of DNA damage signals.

Differential Phosphorylation of FANCA and FANCG Determines Response of Fanconi Anemia Core Complex to DNA Damage and S Phase.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 726-726
Author(s):  
Jun Mi ◽  
Andrei Tomashevski ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Cellular Responses ◽  
S Phase ◽  
Congenital Defects ◽  
Core Complex ◽  
Downstream Target ◽  
Chk1 Kinase ◽  
Differential Phosphorylation

Abstract Fanconi anemia (FA) is a genetic disease marked by bone marrow failure, congenital defects, and cancer. In spite of the identification of at least 8 genes, the biochemistry of the disease and its normal pathway in the cell remains elusive. The FA core complex is composed of at least 5 proteins, 2 of which, FANCA and FANCG, we have shown to be phosphorylated. In these studies, we show that both FANCA and FANCG are phosphorylated in response to DNA damage. In the case of FANCG, we have mapped the site of this phosphorylation to serine 7, using a phosphoserine 7 FANCG antiserum. Because of the link of FA function and the FA core complex-dependent monoubiquitination that occurs both as a result of DNA damage as well as at S phase, we also examined if phosphorylation occurred at S phase as well. While FANCG serine 7 phosphorylation occurs both at S phase and after DNA damage (similar to FANCD2 monoubiquitination), FANCA phosphorylation occurs only after DNA damage. Recent data have implicated the kinase ATR as important in the pathway. In order to assess whether a downstream target of ATR is differentially phosphorylated in FA cells, we tested the phosphorylation status of chk1 in FA-A mutant and corrected cells. Chk1 kinase is phosphorylated at serine 318 in response to DNA damage only in corrected cells but not mutant FA cells, while signaling through chk2 kinase is unaffected. These data suggest the importance of phosphorylation in the FA pathway in the regulation of both cellular responses to DNA damage as well as engagement of the cell cycle.

scholarly journals The E3 Ubiquitin Ligase EDD Regulates S-Phase and G2/M DNA Damage Checkpoints

Cell Cycle ◽  
2007 ◽  
Vol 6 (24) ◽  
pp. 3070-3077 ◽  
Author(s):  
Marcia A. Munoz ◽  
Darren N. Saunders ◽  
Michelle J. Henderson ◽  
Jennifer L. Clancy ◽  
Amanda J. Russell ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Dna Damage Checkpoints

scholarly journals DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

2017 ◽  
Vol 8 (5) ◽  
pp. e2816-e2816 ◽  
Author(s):  
Valérie Glorian ◽  
Jennifer Allègre ◽  
Jean Berthelet ◽  
Baptiste Dumetier ◽  
Pierre-Marie Boutanquoi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Transcriptional Activation ◽  
Cell Cycle Progression ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
E2f Transcription Factor ◽  
Additional Level ◽  
Cell Proliferation Control

Abstract The E2F transcription factor 1 is subtly regulated along the cell cycle progression and in response to DNA damage by post-translational modifications. Here, we demonstrated that the E3-ubiquitin ligase cellular inhibitor of apoptosis 1 (cIAP1) increases E2F1 K63-poly-ubiquitination on the lysine residue 161/164 cluster, which is associated with the transcriptional factor stability and activity. Mutation of these lysine residues completely abrogates the binding of E2F1 to CCNE, TP73 and APAF1 promoters, thus inhibiting transcriptional activation of these genes and E2F1-mediated cell proliferation control. Importantly, E2F1 stabilization in response to etoposide-induced DNA damage or during the S phase of cell cycle, as revealed by cyclin A silencing, is associated with K63-poly-ubiquitinylation of E2F1 on lysine 161/164 residues and involves cIAP1. Our results reveal an additional level of regulation of the stability and the activity of E2F1 by a non-degradative K63-poly-ubiquitination and uncover a novel function for the E3-ubiquitin ligase cIAP1.

scholarly journals Correction: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

2018 ◽  
Vol 9 (8) ◽  
Author(s):  
Valérie Glorian ◽  
Jennifer Allègre ◽  
Jean Berthelet ◽  
Baptiste Dumetier ◽  
Pierre-Marie Boutanquoi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase

scholarly journals Structure of the Fanconi Anemia Core–UBE2T complex poised to ubiquitinate bound FANCI–FANCD2

2019 ◽  
Author(s):  
Shengliu Wang ◽  
Renjing Wang ◽  
Christopher Peralta ◽  
Ayat Yaseen ◽  
Nikola P. Pavletich
Keyword(s):  
Ubiquitin Ligase ◽  
Fanconi Anemia ◽  
Replication Fork ◽  
Single Copy ◽  
Core Complex ◽  
Interstrand Crosslinks ◽  
Ubiquitination Site ◽  
Pathway Activation ◽  
Dna Interstrand Crosslinks ◽  
Ubiquitin Conjugating Enzyme

ABSTRACTThe Fanconi Anemia (FA) pathway is essential for the repair of DNA interstrand crosslinks (ICLs). The pathway is activated when a replication fork stalls because of an ICL or other replication stress. A central event in pathway activation is the mono-ubiquitination of the FANCI-FANCD2 (ID) complex by the FA Core complex, a ubiquitin ligase of nine subunits. Here we describe the cryo-EM structures of the 1.1 MDa FA Core at 3.1 angstroms, except for the FANCA subunit at 3.4, and of the complex containing Core, ID and the UBE2T ubiquitin conjugating enzyme at 4.2 angstroms. The Core has unusual stoichiometry with two copies of FANCB, FAAP100, FANCA, FAAP20, FANCG, FANCL, but only a single copy of FANCC, FANCE and FANCF. This is due to homodimers of FANCA and FANCB having incompatible 2-fold symmetry, resulting in an overall asymmetric assembly of the other subunits. The asymmetry is crucial, as it prevents the binding of a second FANC-C-E-F sub-complex that inhibits UBE2T recruitment by FANCL, and instead creates an ID binding site. The single active FANCL-UBE2T binds next to the FANCD2 ubiquitination site, prying open the FANCI-FANCD2 interface within which the ubiquitination sites are buried. These structures and biochemical data indicate a single active site ubiquitinates FANCD2 and FANCI sequentially, shedding light on a central event in the FA pathway.

Repair pathways independent of the Fanconi anemia nuclear core complex play a predominant role in mitigating formaldehyde-induced DNA damage

2011 ◽  
Vol 404 (1) ◽  
pp. 206-210 ◽  
Author(s):  
Taichi Noda ◽  
Akihisa Takahashi ◽  
Natsuko Kondo ◽  
Eiichiro Mori ◽  
Noritomo Okamoto ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Core Complex ◽  
Predominant Role ◽  
Nuclear Core ◽  
Repair Pathways

scholarly journals Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

2012 ◽  
Vol 23 (21) ◽  
pp. 4203-4211 ◽  
Author(s):  
Dong-Hwan Kim ◽  
Deanna M. Koepp
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Charged Residues ◽  
Phase Progression ◽  
Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

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