Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

Tan, Winnie; Murphy, Vincent J.; Charron, Aude; Twest, Sylvie van; Sharp, Michael F.; Constantinou, Angelos; Parker, Michael W.; Crismani, Wayne; Bythell-Douglas, Rohan; Deans, Andrew J.

doi:10.1371/journal.pone.0229000

Cited by 13 publications

(10 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Human FANCI:FANCD2 complex and Avi-ubiquitin was purified as described in Tan et al (2020). Xenopus laevis (frog) FANCI:FANCD2, human FANCB:FANCL:FAAP100, FANCC:FANCE:FANCF and UBE2T were expressed and purified as described in van .…”

Section: Protein Purificationmentioning

confidence: 99%

ATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2

Tan

Twest

Murphy

et al. 2020

Front. Cell Dev. Biol.

Self Cite

View full text Add to dashboard Cite

DNA interstrand crosslinks (ICLs) are a physical barrier to replication and therefore toxic to cell viability. An important mechanism for the removal of ICLs is the Fanconi Anemia DNA repair pathway, which is initiated by mono-ubiquitination of FANCD2 and its partner protein FANCI. Here, we show that maintenance of FANCD2 and FANCI proteins in a monoubiquitinated form is regulated by the ATR-kinase. Using recombinant proteins in biochemical reconstitution experiments we show that ATR directly phosphorylates FANCI on serine 556, 559, and 565 to stabilize its association with DNA and FANCD2. This increased association with DNA stimulates the conjugation of ubiquitin to both FANCI and FANCD2, but also inhibits ubiquitin deconjugation. Using phosphomimetic and phosphodead mutants of FANCI we show that S559 and S565 are particularly important for protecting the complex from the activity of the deubiquitinating enzyme USP1:UAF1. Our results reveal a major mechanism by which ATR kinase maintains the activation of the FA pathway, by promoting the accumulation of FANCD2 in the ubiquitinated form active in DNA repair.

show abstract

Section: Protein Purificationmentioning

confidence: 99%

ATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2

Tan

Twest

Murphy

et al. 2020

Front. Cell Dev. Biol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…A summary of these proteins is presented in Table 1. Using recombinant ID2 or I Ub D2 Ub prepared by a novel purification method ( Figure 1a) (24) we sought to directly compare the binding of this panel of ID2-associated proteins. Each of the partner proteins was expressed using reticulocyte extracts (Figure 1b), and the majority bound to the ID2 complex as predicted based on previously identified associations ( Figure 1c).…”

Section: Monoubiquitination Does Not Promote Association Of Fanci:fanmentioning

confidence: 99%

“…Di-monoubiquitinated FANCI:FANCD2 complex was purified as described (24). DNA molecules of 60bp or 150bp (dsDNA from oligonucleotides) or 2.6kb (circular plasmid DNA) were used to stimulate the reaction for different experiments, as indicated.…”

Section: Purification Of Monoubiquitinated Fanci:fancd2 Complexmentioning

confidence: 99%

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

Tan

Twest

Leis

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by theFanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This locking of FANCI:FANCD2 complex on DNA requires monoubiquitination of only the FANCD2 subunit. We further show that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA using electron microscopy. Our results reveal how monoubiquitinated FANCI:FANCD2 is activated upon DNA binding and present new insights to potentially modulate monoubiquitinated FANCI:FANCD2/DNA filaments in FA cells. Fanconi Anemia | ubiquitination | DNA repair | biochemistry

show abstract

“…The A627 amino acid is located at the opposite helices facing the protein-protein interface with FANCI that also contains an ubiquitination site at residue K561. FANCD2 ubiquitination stabilizes a conformational change of the protein-protein complex [46] that is required to increase the a nity for the double stranded DNA [47]. The perturbation of the exibility by p.A627S variant of FANCD2 may result in two effects.…”

Section: Discussionmentioning

confidence: 99%

“…The mutation p.A627S is located near a monoubiquitination site on FANCD2 K561 residue (Additional File 3). Ubiquitination can activate the Fanconi anemia DNA repair pathway [46]. This variant induces a signi cant destabilization of the folding energy calculated using DynaMut as well as a perturbation of the local exibility of the structure [47].…”

Section: Molecular Modeling Of the Fancd2 Variantmentioning

confidence: 99%

A rare MSH2 variant as a candidate marker for Lynch Syndrome II screening in Tunisia: A case of Diffuse Gastric Carcinoma

Kabbage¹,

Aissa-Haj²,

Othman³

et al. 2021

Preprint

View full text Add to dashboard Cite

BACKGROUND Inherited syndromic forms of digestive cancers with age occurrence before 50 seem to be relatively in higher proportions in Tunisia suggesting genetic susceptibility. Several syndromic forms are known to predispose to early onset gastric tumors such as Hereditary Diffuse Gastric Cancer (HDGC) and Lynch Syndrome (LS). LS II is an extracolonic cancer syndrome characterized by a tumor spectrum including gastric cancer (GC). In the current work, our main aim was to identify the mutational spectrum underlying the genetic predisposition to diffuse gastric tumors occurring in a Tunisian family suspected with both HDGC and LS II syndromes. PATIENTS AND METHODS The index case was a woman diagnosed with Diffuse Gastric Carcinoma (DGC) fulfilling the international guidelines for both HDGC and LSII syndromes. Genomic DNA was used for the search of CDH1 and CTNNA1 germ mutations. We performed, also a DNA repair custom panel targeting candidate genes recovering the four DNA repair systems. Structural bioinformatics analysis was conducted to predict the effect of the revealed mutations. RESULTS No deleterious variants have been identified neither in CDH1 nor in CTNNA1 coding and their flanking regions. DNA repair genes panel screening identified two variants: a rare MSH2 c.728G > A classified as a variant with uncertain significance and a novel FANCD2 variant c.1879G > T. The structural prediction model of MSH2 mutation and electrostatic potential calculation showed for the first time that MSH2 c.728G > A is likely pathogenic and is involved in the MSH2-MLH1 complex stability. This mutation appears to affect the MSH2-MLH1 complex as well as DNA-complex stability. The c.1879G > T FANCD2 variant was predicted to destabilize the protein structure. CONCLUSIONS Our results showed that MSH2 p.R243Q variant is likely pathogenic and is involved in the MSH2-MLH1 complex stability with a putative impact on the binding with MLH1 by disrupting the electrostatic potential suggesting the revision of its status from VUS to likely pathogenic. This variant seems to be a shared variant in Mediterranean region. These findings emphasize the importance of testing DNA repair genes for patients diagnosed with DGC with suspicion of LSII as well as colorectal cancer allowing better clinical surveillance for more personalized medicine.

show abstract

Preparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin

Cited by 13 publications

References 49 publications

ATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2

ATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

A rare MSH2 variant as a candidate marker for Lynch Syndrome II screening in Tunisia: A case of Diffuse Gastric Carcinoma

Contact Info

Product

Resources

About