title: Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks.
abstract: Fanconi anemia (FA) is a multigene cancer susceptibility disorder characterized by cellular hypersensitivity to DNA interstrand cross-linking agents such as mitomycin C (MMC). FA proteins are suspected to function at the interface between cell cycle checkpoints, DNA repair, and DNA replication. Using replicating extracts from Xenopus eggs, we developed cell-free assays for FA proteins (xFA). Recruitment of the xFA core complex and xFANCD2 to chromatin is strictly dependent on replication initiation, even in the presence of MMC indicating specific recruitment to DNA lesions encountered by the replication machinery. The increase in xFA chromatin binding following treatment with MMC is part of a caffeine-sensitive S-phase checkpoint that is controlled by xATR. Recruitment of xFANCD2, but not xFANCA, is dependent on the xATR-xATR-interacting protein (xATRIP) complex. Immunodepletion of either xFANCA or xFANCD2 from egg extracts results in accumulation of chromosomal DNA breaks during replicative synthesis. Our results suggest coordinated chromatin recruitment of xFA proteins in response to replication-associated DNA lesions and indicate that xFA proteins function to prevent the accumulation of DNA breaks that arise during unperturbed replication.
authors.affiliation: Division of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, Oregon 97239, USA.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Sobeck
authors.forename: Stacie
authors.initials: S
authors.lastname: Stone
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Costanzo
authors.forename: Bendert
authors.initials: B
authors.lastname: de Graaf
authors.forename: Tanja
authors.initials: T
authors.lastname: Reuter
authors.forename: Johan
authors.initials: J
authors.lastname: de Winter
authors.forename: Michael
authors.initials: M
authors.lastname: Wallisch
authors.forename: Yassmine
authors.initials: Y
authors.lastname: Akkari
authors.forename: Susan
authors.initials: S
authors.lastname: Olson
authors.forename: Weidong
authors.initials: W
authors.lastname: Wang
authors.forename: Hans
authors.initials: H
authors.lastname: Joenje
authors.forename: Jan L
authors.initials: JL
authors.lastname: Christian
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Lupardus
authors.forename: Karlene A
authors.initials: KA
authors.lastname: Cimprich
authors.forename: Jean
authors.initials: J
authors.lastname: Gautier
authors.forename: Maureen E
authors.initials: ME
authors.lastname: Hoatlin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004249:DNA Damage / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology; D004261:DNA Replication; D052217:Fanconi Anemia Complementation Group A Protein / Q000378:metabolism*; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000378:metabolism*; D051856:Fanconi Anemia Complementation Group Proteins / Q000378:metabolism*; D005260:Female; D066298:In Vitro Techniques; D016685:Mitomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016196:S Phase / Q000187:drug effects / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: C495984:ATRIP protein, Xenopus; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D003432:Cross-Linking Reagents; C576817:FANCD2 protein, Xenopus; D052217:Fanconi Anemia Complementation Group A Protein; D052236:Fanconi Anemia Complementation Group D2 Protein; D051856:Fanconi Anemia Complementation Group Proteins; D029867:Xenopus Proteins; D002110:Caffeine; D016685:Mitomycin; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases