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The structure of the catalytic subunit FANCL of the Fanconi anemia core complex.

ABSTRACT: The Fanconi anemia (FA) pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the FA core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the FA core complex, at 3.2 A. The structure reveals an architecture fundamentally different from previous sequence-based predictions. The molecule is composed of an N-terminal E2-like fold, which we term the ELF domain, a novel double-RWD (DRWD) domain, and a C-terminal really interesting new gene (RING) domain predicted to facilitate E2 binding. Binding assays show that the DRWD domain, but not the ELF domain, is responsible for substrate binding.

SUBMITTER: Cole AR

PROVIDER: S-EPMC2929457 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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The structure of the catalytic subunit FANCL of the Fanconi anemia core complex.

Cole Ambrose R AR Lewis Laurence P C LP Walden Helen H

Nature structural & molecular biology 20100214 3

The Fanconi anemia (FA) pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the FA core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the FA core complex, at 3.2 A. The structure reveals an architecture fundamentally different from previous sequence-based predictions. The molecule is composed of an N-terminal E2-like fold, which we term the ELF domain, a novel double-RWD (DRWD) domain, and a C-term ...[more]

PMID: 20154706

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Modularized functions of the Fanconi anemia core complex.

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The Fanconi anemia core complex is required for efficient point mutagenesis and Rev1 foci assembly.

Project description:Fanconi anemia (FA) is a chromosome instability syndrome characterized by congenital abnormalities, cellular hypersensitivity to DNA crosslinking agents, and heightened cancer risk. Eight of the thirteen identified FA genes encode subunits of a nuclear FA core complex that monoubiquitinates FANCD2 and FANCI to maintain genomic stability in response to replication stress. The FA pathway has been implicated in the regulation of error-prone DNA damage tolerance via an undefined molecular mechanism. Here, we show that the FA core complex is required for efficient spontaneous and UVC-induced point mutagenesis, independently of FANCD2 and FANCI. Consistent with the observed hypomutability of cells deficient in the FA core complex, we also demonstrate that these cells are impaired in the assembly of the error-prone translesion DNA synthesis polymerase Rev1 into nuclear foci. Consistent with a role downstream of the FA core complex and like known FA proteins, Rev1 is required to prevent DNA crosslinker-induced chromosomal aberrations in human cells. Interestingly, proliferating cell nuclear antigen (PCNA) monoubiquitination, known to contribute to Rev1 recruitment, does not require FA core complex function. Our results suggest a role for the FA core complex in regulating Rev1-dependent DNA damage tolerance independently of FANCD2, FANCI, and PCNA monoubiquitination.

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FANCM of the Fanconi anemia core complex is required for both monoubiquitination and DNA repair.

Project description:In response to DNA damage, the Fanconi anemia (FA) core complex functions as a signaling machine for monoubiquitination of FANCD2 and FANCI. It remains unclear whether this complex can also participate in subsequent DNA repair. We have shown previously that the FANCM constituent of the complex contains a highly conserved helicase domain and an associated ATP-dependent DNA translocase activity. Here we show that FANCM also possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimics intermediates generated during homologous recombination or at stalled replication forks. Using an siRNA-based complementation system, we found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA-crosslinking drug, mitomycin C, but not for the monoubiquitination of FANCD2 and FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair.

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Monoubiquitination by the human Fanconi anemia core complex clamps FANCI:FANCD2 on DNA in filamentous arrays.

Project description:FANCI:FANCD2 monoubiquitination is a critical event for replication fork stabilization by the Fanconi anemia (FA) DNA repair pathway. It has been proposed that at stalled replication forks, monoubiquitinated-FANCD2 serves to recruit DNA repair proteins that contain ubiquitin-binding motifs. Here, we have reconstituted the FA pathway in vitro to study functional consequences of FANCI:FANCD2 monoubiquitination. We report that monoubiquitination does not promote any specific exogenous protein:protein interactions, but instead stabilizes FANCI:FANCD2 heterodimers on dsDNA. This clamping requires monoubiquitination of only the FANCD2 subunit. We further show using electron microscopy that purified monoubiquitinated FANCI:FANCD2 forms filament-like arrays on long dsDNA. Our results reveal how monoubiquitinated FANCI:FANCD2, defective in many cancer types and all cases of FA, is activated upon DNA binding.

| S-EPMC7156235 | biostudies-literature

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