A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

Song, Ihn Young; Barkley, Laura R.; Day, Tovah; Weiss, Robert S.; Vaziri, Cyrus

doi:10.4161/cc.9.12.11900

Cited by 20 publications

(17 citation statements)

References 45 publications

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“…6, we observed slightly reduced PCNA ubiquitylation in FANCD2-and FANCI-depleted H1299 cells relative to controls. However, we have found that FANCD2 deficiency results in reduced origin firing in many cultured cell lines, including H1299 (38). Therefore, the attenuated PCNA ubiquitylation observed following FANCD2/FANCI depletion likely results from reduced numbers of replicons in these cells.…”

Section: Role Of Rad18-mediated Pcna Mono-ubiquitination In Famentioning

confidence: 91%

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Song

Palle

Gurkar

et al. 2010

Journal of Biological Chemistry

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Fanconi anemia (FA) is a cancer susceptibility syndrome characterized by sensitivity to DNA-damaging agents. The FA proteins (FANCs) are implicated in DNA repair, although the precise mechanisms by which FANCs process DNA lesions are not fully understood. An epistatic relationship between the FA pathway and translesion synthesis (TLS, a post-replication DNA repair mechanism) has been suggested, but the basis for crosstalk between the FA and TLS pathways is poorly understood. We show here that ectopic overexpression of the E3 ubiquitin ligase Rad18 (a central regulator of TLS) induces DNA damage-independent mono-ubiquitination of proliferating cell nuclear antigen (PCNA) (a known Rad18 substrate) and FANCD2. Conversely, DNA damage-induced mono-ubiquitination of both PCNA and FANCD2 is attenuated in Rad18-deficient cells, demonstrating that Rad18 contributes to activation of the FA pathway. WT Rad18 but not an E3 ubiquitin ligase-deficient Rad18 C28F mutant fully complements both PCNA ubiquitination and FANCD2 activation in Rad18-depleted cells. Rad18-induced mono-ubiquitination of FANCD2 is not observed in FA core complex-deficient cells, demonstrating that Rad18 E3 ligase activity alone is insufficient for FANCD2 ubiquitylation. Instead, Rad18 promotes FA core complex-dependent FANCD2 ubiquitination in a manner that is secondary to PCNA monoubiquitination. Taken together, these results demonstrate a novel Rad18-dependent mechanism that couples activation of the FA pathway with TLS.

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Section: Role Of Rad18-mediated Pcna Mono-ubiquitination In Famentioning

confidence: 91%

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Song

Palle

Gurkar

et al. 2010

Journal of Biological Chemistry

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“…After washing extensively with PBST to remove unbound secondary antibody, the slides were for FANC status by immunoblotting as described in recent publications. 45,64,65 All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, streptomycin sulfate (100 μg/ml) and penicillin (100 U/ml), 5 mM L-glutamine, and cultures were incubated at 37°C in humidified chambers with 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

Palle¹,

Vaziri²

2011

Cell Cycle

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Camptothecin (Cpt) and related chemotherapeutic drugs induce formation of DNA topoisomerase I (top1) covalent or cleavage complexes (top1ccs) that block leading-strand DNA synthesis and elicit DNA Double Stranded Breaks (DSB) during S phase. the Fanconi Anemia (FA) pathway is implicated in tolerance of Cpt-induced DNA damage yet the mechanism of FA pathway activation by top1 poisons has not been studied. We show here that the FA core complex protein FANCA and monoubiquitinated FANCD2 (an effector of the FA pathway) are rapidly mobilized to chromatin in response to Cpt treatment in several human cancer cell lines and untransformed primary human dermal fibroblasts. FANCD2 depletion using siRNA leads to impaired recovery from Cpt-induced inhibition or DNA synthesis, persistence of γH2AX (a DSB marker) and reduced cell survival following Cpt treatment. the e3 ubiquitin ligase Rad18 is necessary for Cpt-induced recruitment of FANCA and FANCD2 to chromatin. Moreover, Rad18-depletion recapitulates the DNA synthesis and survival defects of FANCD2-deficiency in Cpt-treated cells. It is well-established that Rad18 promotes FA pathway activation and DNA damage tolerance in response to bulky DNA lesions via a mechanism involving pCNA monoubiquitination. In contrast, pCNA monoubiquitination is not involved in Rad18-mediated FA pathway activation or cell survival following acquisition of Cpt-induced DSB. Moreover, while Rad18 is implicated in recombinational repair of DSB via an e3 ligase-independent mechanism, we demonstrate that Rad18 e3 ligase activity is essential for appropriate FA pathway activation and DNA damage tolerance after Cpt treatment. taken together, our results define a novel pathway of Rad18-dependent DSB repair that is dissociable from known Rad18-mediated DNA repair mechanisms based on its independence from pCNA ubiquitination and requirement for e3 ligase activity. MMS, methyl methanesulfonate; NBS1, nijmegen breakage syndrome protein1; NHEJ, non-homologous end joining; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; Polη, DNA polymerases eta; Polι, DNA polymerases iota; Polκ, DNA polymerases kappa; RFC, replication factor C; RPA, replication protein A; RT, room temperature; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; siRNA, small interference RNA; ssDNA, single-stranded DNA; TBE, tris-boric acid-EDTA (buffer); TLS, translesion DNA synthesis; Top1, DNA topoisomerase I; Top1cc, DNA-topoisomerase I covalent or cleavage complex; Usp1, ubiquitin-specific processing protease 1; UV, ultra violet radiation; WT, wild type ©2 0 1 1 L a n d e s B i o s c i e n c e . D o n o t d i s t r i b u t e .1626 Cell Cycle Volume 10 Issue 10 with TLS, ensuring appropriate processing of bulky DNA lesions. FA is a cancer predisposition syndrome in humans. To date, at least 13 complementation groups of FA have been identified (designated 'FANC' A-M). The FA proteins (FANCs) are thought to work cooperatively in a common pathway to repair interstrand crosslinks. 28 The eight-prote...

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“…112 In addition, the Vaziri group has recently shown that FANCD2 is necessary for efficient initiation of DNA replication in primary human fibroblasts. 113 An unbiased proteomics screen of chromatinbound FANCD2 immune complexes revealed that FANCD2 physically associates with the minichromosome maintenance 2-7 (MCM2-MCM7) replicative helicases. 114,115 FANCD2 interacts with MCM2-MCM7 in response to ATR-mediated DNA replication stress signaling independently of the core FA complex and monoubiquitination.…”

Section: Fancd2 and Fanci Functionmentioning

confidence: 99%

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

Boisvert

Howlett

2014

Cell Cycle

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A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

Cited by 20 publications

References 45 publications

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Rad18-mediated Translesion Synthesis of Bulky DNA Adducts Is Coupled to Activation of the Fanconi Anemia DNA Repair Pathway

Rad18 E3 ubiquitin ligase activity mediates Fanconi anemia pathway activation and cell survival following DNA Topoisomerase 1 inhibition

The Fanconi anemia ID2 complex: Dueling saxes at the crossroads

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