DNA structure-induced recruitment and activation of the Fanconi anemia pathway protein FANCD2.
The Fanconi anemia (FA) pathway proteins are thought to be involved in the repair of irregular DNA structures including those encountered by the moving replication fork. However, the nature of the DNA structures that recruit and activate the FA proteins is not known. Because FA proteins function within an extended network of proteins, some of which are still unknown, we recently established cell-free assays in Xenopus laevis egg extracts to deconstruct the FA pathway in a fully replication-competent context. Here we show that the central FA pathway protein, xFANCD2, is monoubiquitinated (xFANCD2-L) rapidly in the presence of linear and branched double-stranded DNA (dsDNA) structures but not single-stranded or Y-shaped DNA. xFANCD2-L associates with dsDNA structures in an FA core complex-dependent manner but independently of xATRIP, the regulatory subunit of xATR. Formation of xFANCD2-L is also triggered in response to circular dsDNA, suggesting that dsDNA ends are not required to trigger monoubiquitination of FANCD2. The induction of xFANCD2-L in response to circular dsDNA is replication and checkpoint independent. Our results provide new evidence that the FA pathway discriminates among DNA structures and demonstrate that triggering the FA pathway can be uncoupled from DNA replication and ATRIP-dependent activation.
Sobeck
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Biochemistry and Molecular Biology and Molecular and Medical Genetics, Oregon Health and Science University, Medical Research Building, Portland, OR 97239, USA.
Stone
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Hoatlin
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D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry*; D004261:DNA Replication; D004270:DNA, Circular / Q000737:chemistry*; D018076:DNA, Complementary; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D005260:Female; D009865:Oocytes / Q000737:chemistry; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D025801:Ubiquitin / Q000032:analysis / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
C495984:ATRIP protein, Xenopus; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004270:DNA, Circular; D018076:DNA, Complementary; D052236:Fanconi Anemia Complementation Group D2 Protein; D025801:Ubiquitin; D029867:Xenopus Proteins; D004247:DNA; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases