FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response

Abstract

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2—but not FANCI—plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response.

Monoubiquitination by the Fanconi Anemia core complex locks FANCI:FANCD2 on DNA in filamentous arrays

“…Previous studies reported that monoubiquitination of ID2 complex may lead to dissociation of the heterodimer to its individual subunits, as measured by loss of co-immunoprecipitation of FANCI with FANCD2 (35,36). In contrast, we did not observe any Ub-mediated dissociation of ID2 in vitro.…”

Dysfunctional DNA repair pathway via defective FANCD2 gene engenders multifarious exomic and transcriptomic effects in Fanconi anemia

“…Unlike SNPs, INDELs were more prevalent in FA than FA_RV samples, suggesting that INDELs may have continued to accumulate in the FANCD2 ‐uncorrected cell lines. FANCD2 deficiency is related to replication fork restart defects (Thompson et al, ), and stalled replication forks have been known to result in INDELs (Sankar, Wastuwidyaningtyas, Dong, Lewis, & Wang, ). However, it should be noted that this analysis does not rule out effects of other genome repair checkpoints or even side effects of cell immortalization.…”

Bi-allelic MCM10 mutations cause telomere shortening with immune dysfunction and cardiomyopathy

“…Cell Line Generation using rAAV HCT116 MCM10 +/-(exon 14) cell lines were generated using rAAV (recombinant adenoassociated virus)-mediated gene targeting (Kohli et al, 2004). The conditional vector pAAV-MCM10-cond was constructed using Golden Gate cloning and designed as described previously (Kohli et al, 2004, Thompson et al, 2017. The MCM10-cond rAAV was generated by co-transfection of pAAV-MCM10-cond, pAAV-RC (Kohli et al, 2004), and pHelper (Kohli et al, 2004) into HEK293 cells using Lipofectamine LTX (Invitrogen 15338030) following standard protocols (Kohli et al, 2004).…”

“…Targeted clones were selected using 0.5 mg/ml G418 (Geneticin G5005). Resistant clones were screened by PCR using primers within the neomycin cassette and outside the rAAV homology arms to confirm locus-specific targeting, and Cre (cyclization recombinase) transiently expressed from an adenoviral vector (AdCre; Vector Biolabs #1045) was then used to remove the neomycin selection cassette as described (Kohli et al, 2004, Thompson et al, 2017. The second round of MCM10 gene targeting used the same rAAV vector and replaced the wild type allele with a floxed allele and a downstream floxed neomycin selection cassette.…”