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ItemATR-Mediated FANCI Phosphorylation Regulates Both Ubiquitination and Deubiquitination of FANCD2(Frontiers Media, 2020-02-04)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.
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ItemATP-dependent helicase activity is dispensable for the physiological functions of Recql4(PUBLIC LIBRARY SCIENCE, 2019-07)Rothmund-Thomson syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash (poikiloderma), skeletal dysplasia, small stature, juvenile cataracts, sparse or absent hair, and predisposition to specific malignancies such as osteosarcoma and hematological neoplasms. RTS is caused by germ-line mutations in RECQL4, a RecQ helicase family member. In vitro studies have identified functions for the ATP-dependent helicase of RECQL4. However, its specific role in vivo remains unclear. To determine the physiological requirement and the biological functions of Recql4 helicase activity, we generated mice with an ATP-binding-deficient knock-in mutation (Recql4K525A). Recql4K525A/K525A mice were strikingly normal in terms of embryonic development, body weight, hematopoiesis, B and T cell development, and physiological DNA damage repair. However, mice bearing two distinct truncating mutations Recql4G522Efs and Recql4R347*, that abolished not only the helicase but also the C-terminal domain, developed a profound bone marrow failure and decrease in survival similar to a Recql4 null allele. These results demonstrate that the ATP-dependent helicase activity of Recql4 is not essential for its physiological functions and that other domains might contribute to this phenotype. Future studies need to be performed to elucidate the complex interactions of RECQL4 domains and its contribution to the development of RTS.
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ItemPreparation and purification of mono-ubiquitinated proteins using Avi-tagged ubiquitin(PUBLIC LIBRARY SCIENCE, 2020-02-24)Site-specific conjugation of ubiquitin onto a range of DNA repair proteins regulates their critical functions in the DNA damage response. Biochemical and structural characterization of these functions are limited by an absence of tools for the purification of DNA repair proteins in purely the ubiquitinated form. To overcome this barrier, we designed a ubiquitin fusion protein that is N-terminally biotinylated and can be conjugated by E3 RING ligases onto various substrates. Biotin affinity purification of modified proteins, followed by cleavage of the affinity tag leads to release of natively-mono-ubiquitinated substrates. As proof-of-principle, we applied this method to several substrates of mono-ubiquitination in the Fanconi anemia (FA)-BRCA pathway of DNA interstrand crosslink repair. These include the FANCI:FANCD2 complex, the PCNA trimer and BRCA1 modified nucleosomes. This method provides a simple approach to study the role of mono-ubiquitination in DNA repair or any other mono-ubiquitination signaling pathways.