Impaired FANCD2 monoubiquitination and hypersensitivity to camptothecin uniquely characterize Fanconi anemia complementation group M

Abstract

FANCM is a component of the Fanconi anemia (FA) core complex and one FA patient (EUFA867) with biallelic mutations in FANCM has been described. Strikingly, we found that EUFA867 also carries biallelic mutations in FANCA. After correcting the FANCA defect in EUFA867 lymphoblasts, a "clean" FA-M cell line was generated. These cells were hypersensitive to mitomycin C, but unlike cells defective in other core complex members, FANCM Ϫ/Ϫ cells were proficient in monoubiquitinating FANCD2 and were sensitive to the topoisomerase inhibitor camptothecin, a feature shared only with the FA subtype D1 and N. In addition, FANCM Ϫ/Ϫ cells were sensitive to UV light. IntroductionFanconi anemia (FA) is a recessive genetic instability syndrome that has uncovered a cellular pathway involved in the protection against replication-blocking lesions. Inactivation of this pathway, as seen in FA patients, results in hypersensitivity to DNA crosslinking agents and cancer susceptibility. 1 Defects in 13 different genes have been found in FA patients, 2 and the proteins encoded by these genes cooperate in a pathway that can be subdivided in an upstream and downstream part based upon the monoubiquitination of FANCD2 and FANCI. 1 The upstream part of the pathway consists of a nuclear core complex formed by the FA proteins FANCA, -B, -C, -E, -F, -G, -L, and -M and 2 FA-associated proteins FAAP100 and FAAP24. This complex monoubiquitinates FANCD2 through the E3-ubiquitin ligase FANCL in conjunction with the ubiquitin-conjugating enzyme. 3,4 The FA core complex, UBE2T, and FANCD2 are independently recruited to the stalled replication fork. 5 For FANCD2, this relies on the ATR-mediated phosphorylation of its binding partner FANCI, 6 whereas the recruitment of the FA core complex seems to depend on FANCM. 7 Like FANCD2, FANCI is also monoubiquitinated by the FA core complex and these modified proteins colocalize with Rad51 and BRCA1 in nuclear foci. 8,9 The link between FA and BRCA proteins was further strengthened by the discovery of FA patients with mutations in BRCA2, 10 and in the BRCA1-and BRCA2-interacting proteins BRIP1 11,12 and PALB2. 13,14 FA patients with a defect in any of these genes have normal FANCD2 monoubiquitination and therefore these proteins are considered as downstream players in the FA pathway.Despite the identification of the various components of the FA core complex, its role in the maintenance of genome stability remains unclear, because of the absence of functional domains in most of the core complex members. A notable exception is FANCM, an ortholog of the archaeal DNA repair protein HEF, which contains 2 conserved domains: a DEAH helicase domain in the N-terminus and an endonuclease domain in the C-terminus. 15,16 The helicase domain is shared with yeast orthologs MPH1 (Saccharomyces cerevisiae) and FML1 (Schizosaccharomyces pombe), which play a regulatory role in homologous recombination repair by replication fork reversal and D-loop disruption. [17][18][19] HEF and MPH1 possess helicase activity, 20,21 whereas for F...

Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer

“…Also, in colorectal cancer (CRC) patients, an FANCM nonsense mutation (c.5791C>T, p.Arg1931Ter) has been identified (37), but further studies are needed to clarify the potential role of FANCM in CRC susceptibility. However, only one FA patient has been found to carry truncating FANCM mutation and this individual also carries biallelic mutations in the FANCA gene (33,38). Furthermore, homozygous carriers of FANCM loss-of-function mutations c.5101C>T and c.5791C>T observed in the Finnish population do not show symptoms of FA (39).…”

Cytokinesis failure occurs in Fanconi anemia pathway–deficient murine and human bone marrow hematopoietic cells

“…Complementation of the shFANCM cell line with a siRNA-resistant WT FANCM rescued the defect ( Figure 2F). FANCM plays at least 2 independent functions in the FA pathway: first, it recruits the core complex to chromatin and promotes FANCD2-I ubiquitination; second, it plays a more direct role in repair (22). Accordingly, a patient-derived FANCM-deficient cell line is partially deficient for FANCD2 ubiquitination and displays MMC sensitivity (22).…”

Defects in homologous recombination repair behind the human diseases: FA and HBOC

“…Biallelic FANCM mutations were identified in a single patient, but the defects in cells from the patient could not be reversed by the expression of wild-type FANCM (Meetei et al 2005). This was later found to be due to the presence of simultaneous mutations in FANCA (Singh et al 2009). Thus, to date, there have been no human FA patients identified with causative mutations solely in FANCM.…”