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Cell Rep. 2019 Jan 15;26(3):564-572.e5. doi: 10.1016/j.celrep.2018.12.084.

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

Author information

1
Department of Pediatrics, Yale Medical School, New Haven, CT 06520, USA; Department of Molecular Biology and Biophysics, Yale Medical School, New Haven, CT 06520, USA.
2
School of Medicine, Tsinghua University, No.1 Tsinghua Yuan, Haidian District, Beijing 100084, China; Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.
3
Department of Pediatrics, Yale Medical School, New Haven, CT 06520, USA.
4
Department of Molecular Biology and Biophysics, Yale Medical School, New Haven, CT 06520, USA.
5
Department of Pediatrics, Yale Medical School, New Haven, CT 06520, USA; Department of Pathology, Yale Medical School, New Haven, CT 06520, USA.
6
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
7
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA; Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA 02129, USA; Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129, USA.
8
Department of Molecular Biology and Biophysics, Yale Medical School, New Haven, CT 06520, USA; Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA. Electronic address: sungp@uthscsa.edu.
9
Department of Pediatrics, Yale Medical School, New Haven, CT 06520, USA; Department of Pathology, Yale Medical School, New Haven, CT 06520, USA. Electronic address: gary.kupfer@yale.edu.

Abstract

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.

KEYWORDS:

DNA damage; FANCD2; FANCI-FANCD2; Fanconi anemia; R-loop; RNA; monoubiquitination; transcription perturbation

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