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Cryo-EM movies of the Gallus gallus FANCD2-FANCI in complex with dsDNA

ABSTRACT:

SUBMITTER: Pablo Alcón

PROVIDER: EMPIAR-12394 | biostudies-other |

REPOSITORIES: biostudies-other

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FANCD2-FANCI surveys DNA and recognizes double- to single-stranded junctions.

Alcón Pablo P Kaczmarczyk Artur P AP Ray Korak Kumar KK Liolios Themistoklis T Guilbaud Guillaume G Sijacki Tamara T Shen Yichao Y McLaughlin Stephen H SH Sale Julian E JE Knipscheer Puck P Rueda David S DS Passmore Lori A LA

Nature 20240731 8027

DNA crosslinks block DNA replication and are repaired by the Fanconi anaemia pathway. The FANCD2-FANCI (D2-I) protein complex is central to this process as it initiates repair by coordinating DNA incisions around the lesion<sup>1</sup>. However, D2-I is also known to have a more general role in DNA repair and in protecting stalled replication forks from unscheduled degradation<sup>2-4</sup>. At present, it is unclear how DNA crosslinks are recognized and how D2-I functions in replication fork pr ...[more]

PMID: 39085614

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Cryo-EM movies of the Gallus gallus FANCD2-FANCI in complex with ss-dsDNA

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Binding of FANCI-FANCD2 Complex to RNA and R-Loops Stimulates Robust FANCD2 Monoubiquitination.

Project description:Fanconi anemia (FA) is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. FA cells are hypersensitive to DNA replicative stress and accumulate co-transcriptional R-loops. Here, we use the Damage At RNA Transcription assay to reveal colocalization of FANCD2 with R-loops in a highly transcribed genomic locus upon DNA damage. We further demonstrate that highly purified human FANCI-FANCD2 (ID2) complex binds synthetic single-stranded RNA (ssRNA) and R-loop substrates with high affinity, preferring guanine-rich sequences. Importantly, we elucidate that human ID2 binds an R-loop structure via recognition of the displaced ssDNA and ssRNA but not the RNA:DNA hybrids. Finally, a series of RNA and R-loop substrates are found to strongly stimulate ID2 monoubiquitination, with activity corresponding to their binding affinity. In summary, our results support a mechanism whereby the ID2 complex suppresses the formation of pathogenic R-loops by binding ssRNA and ssDNA species, thereby activating the FA pathway.

| S-EPMC6350941 | biostudies-literature

The FANCD2-FANCI complex is recruited to DNA interstrand crosslinks before monoubiquitination of FANCD2.

Project description:The Fanconi anaemia (FA) pathway is important for the repair of DNA interstrand crosslinks (ICL). The FANCD2-FANCI complex is central to the pathway, and localizes to ICLs dependent on its monoubiquitination. It has remained elusive whether the complex is recruited before or after the critical monoubiquitination. Here, we report the first structural insight into the human FANCD2-FANCI complex by obtaining the cryo-EM structure. The complex contains an inner cavity, large enough to accommodate a double-stranded DNA helix, as well as a protruding Tower domain. Disease-causing mutations in the Tower domain are observed in several FA patients. Our work reveals that recruitment of the complex to a stalled replication fork serves as the trigger for the activating monoubiquitination event. Taken together, our results uncover the mechanism of how the FANCD2-FANCI complex activates the FA pathway, and explains the underlying molecular defect in FA patients with mutations in the Tower domain.

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Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA.

Project description:The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues. However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2's affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary "Arm" ID2 interface. Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.

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Structural insight into FANCI-FANCD2 monoubiquitination.

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FANCD2 regulates BLM complex functions independently of FANCI to promote replication fork recovery.

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Ubiquitinated FANCI in complex with FANCD2 and double-stranded DNA

Project description: Not available

| EMPIAR-11372 | biostudies-other

Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage.

Project description:Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.

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Structure of the FANCI-FANCD2 complex: insights into the Fanconi anemia DNA repair pathway.

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