Fanconi anemia proteins are required to prevent accumulation of replication-associated DNA double-strand breaks.
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.
Sobeck
Alexandra
A
Division of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd., Portland, Oregon 97239, USA.
Stone
Stacie
S
Costanzo
Vincenzo
V
de Graaf
Bendert
B
Reuter
Tanja
T
de Winter
Johan
J
Wallisch
Michael
M
Akkari
Yassmine
Y
Olson
Susan
S
Wang
Weidong
W
Joenje
Hans
H
Christian
Jan L
JL
Lupardus
Patrick J
PJ
Cimprich
Karlene A
KA
Gautier
Jean
J
Hoatlin
Maureen E
ME
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
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