Fig 2: RNA and R-Loop Stimulate FANCD2 Monoubiquitination(A) In vitro ubiquitination reaction of recombinant ID2 with either ssDNA or ssRNA substrates. FANCD2 was ubiquitinated maximally in presence of random sequence of ssRNA or ssDNA and ubiquitination was abrogated by T or U-rich nucleic acids.(B) DNA and RNA binding mutant FANCI(KKEE)/D2 complex failed to be ubiquitinated.(C) Comparison of ID2 ubiquitination efficiency using various nucleic acids substrates with different binding affinity. ssRNA conferred the most ubiquitination.(D) Comparison of ID2 ubiquitination efficiency using nucleic acids substrates with different guanine content. Nucleic acid with the highest guanine content conferred the most ubiquitination.(E) Comparison of ID2 ubiquitination efficiency using two configurations of D-loops and R-loops. D- and R-loops with guanine-containing single-strand DNA (ssDNA) supported ID2 ubiquitination similarly.(F) Comparison of ID2 ubiquitination efficiency using different R-loop substrates. R-loops with guanine-containing ssDNA and ssRNA tail supported ID2 ubiquitination with the highest efficiency. For (C)–(F), percentage of ubiquitinated FANCD2 is calculated by the intensity of the Ub-D2 band over total intensity of the Ub-D2 and D2 bands.(G) Model of R-loop binding and its stimulated monoubiquitination of FANCI-FANCD2 in response to DNA damage-induced transcriptional perturbation.

Fig 3: FANCD2 and FANCI are required for HIV-1 DNA integration(A–J) FANCD2 (A–E) and FANCI (F–J) are required for HIV-1 DNA integration. Indicated cells were infected with VSV-G pseudotyped HIV-Luc virus. The expression of indicated proteins was determined by western blot (A and F). Luciferase activities (B and C), total viral DNA (C and H), unintegrated viral DNA (D and I), and integrated viral DNA (E and J) were measured at indicated time points post-infection. Data are represented as means ± SDs from 3 independent experiments (n = 3). ns p > 0.05; *p < 0.05; **p < 0.01.(K and L) Knockdown of FANCI inhibits the replication of HIV-1. MT-4 cells stably expressing control shRNA (SCR) or shRNAs targeting FANCI were infected with HIV-1(NL4.3) at 1 ng CA/106 cells. The expression of FANCI was determined by western blot (K). Viral spreading was monitored by assay for the infectivity of supernatants in TZM-bl cells engineered with the HIV-1 Tat-responsive luciferase reporter gene (L). Data presented are means ± SEMs from 2 technical replicates and are representative of 2 independent experiments.See also Figure S2.

Fig 4: Fanci−/− animals are smaller and show eye defects. (A) Embryonic lethality of Fanci−/− mice. Genotyping of the litters from heterozygous mating (n = 386 mice genotyped). (B and C) Fanci−/− mice are smaller. (B) Representative picture of Fanci+/+ and Fanci−/− new born pups, illustrating the growth delay at birth and 2-weeks-old. Statistical analysis Mann–Whitney (ns: P> 0.05, *P< 0.05, **P< 0.01, ***P< 0.001). (C) Animal weights of each genotype were followed for 10 weeks. KO males had a 20% growth delay (P < 0.01) compared to the weight of WT males, and females showed a 30% delay (P < 0.005). (D) Tomography of two males mice from the same litter at 4 months. The Fanci−/− mice are smaller but do not show missing vertebraes. (E) Fanci−/− mice have eyes defects. Representative picture of the heads of mice showing anophtalmia, opaque cornea, or impaired development of the lens (Fisher exact test ****P< 0.0001).

Fig 5: FANCD2 and FANCI interact with RAD51. (A) Pull-down assay with the HeLa cell extracts. The beads without RAD51 (N) or with RAD51 (R) were incubated with the nuclease-treated cell extracts, and the proteins bound to the beads were detected by Western blotting. The cell extracts were prepared from untreated and MMC-treated HeLa cells. (B) Pull-down assay with the DT40 cell extracts. The nuclease-treated cell extracts were prepared from untreated, MMC-treated and HU-treated DT40 cells. (C) Pull-down assay with Ni-NTA beads. RAD51 bound to His6-tagged FANCI, FANCD2 and the I-D complex was copelleted with the Ni-NTA beads, and the proteins were analyzed by SDS-PAGE. (D) Pull-down assay with single-stranded DNA (ssDNA) beads. The beads conjugated with ssDNA, the RAD51-ssDNA complex or the RPA-ssDNA complex were incubated with the I-D complex. The I-D complex that copelleted with the beads was analyzed by SDS-PAGE. The amounts of the I-D complex in the bound fractions were quantitated, and the mean percentages of three independent experiments are indicated with the standard deviations. (E) Pull-down assay with His6-tagged RAD51 proteins. The I-D complex was copelleted with His6-RAD51 or His6-RAD51 F86E bound to Ni-NTA beads, and the proteins were analyzed by SDS-PAGE with Coomassie Brilliant Blue staining. The amounts of the I-D complex in the bound fractions were quantitated, and the mean percentages of three independent experiments are indicated with the standard deviations.