The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange

Abstract

Fanconi anemia (FA) is a human autosomal disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinking agents such as mitomycin C and diepoxybutane. Six FA genes have been cloned including a gene designated XRCC9 (for X-ray Repair Cross Complementing), isolated using a mitomycin C-hypersensitive Chinese hamster cell mutant termed UV40, and subsequently found to be identical to FANCG. A nuclear complex containing the FANCA, FANCC, FANCE, FANCF and FANCG proteins is needed for the activation of a sixth FA protein FANCD2. When monoubiquitinated, the FANCD2 protein co-localizes with the breast cancer susceptibility protein BRCA1 in DNA damage induced foci. In this study, we have assigned NM3, a nitrogen mustard-hypersensitive Chinese hamster mutant to the same genetic complementation group as UV40. NM3, like human FA cell lines (but unlike UV40) exhibits a normal spontaneous level of sister chromatid exchange. We show that both NM3 and UV40 are also hypersensitive to other DNA crosslinking agents (including diepoxybutane and chlorambucil) and to non-crosslinking DNA damaging agents (including bleomycin, streptonigrin and EMS), and that all these sensitivities are all corrected upon transfection of the human FANCG/XRCC9 cDNA. Using immunoblotting, NM3 and UV40 were found not to express the active monoubiquitinated isoform of the FANCD2 protein, although expression of the FANCD-L isoform was restored in the FANCG cDNA transformants, correlating with the correction of mutagen-sensitivity. These data indicate that cellular resistance to these DNA damaging agents requires FANCG and that the FA gene pathway, via its activation of FANCD2 and that protein's subsequent interaction with BRCA1, is involved in maintaining genomic stability in response not only to DNA interstrand crosslinks but also a range of other DNA damages including DNA strand breaks. NM3 and other "FA-like" Chinese hamster mutants should provide an important resource for the study of these processes in mammalian cells.

Phosphorylation of Fanconi Anemia (FA) Complementation Group G Protein, FANCG, at Serine 7 Is Important for Function of the FA Pathway

“…NM3 and UV40 both contain frameshift mutations that cause premature truncation of FANCG in exons 3 and 1, respectively, and fail to express FANCG protein (59). Wild-type human FANCG corrected the MMC sensitivity phenotype of the NM3 (D 37 ϭ 120 nM versus D 37 ϭ 28 nM for vector only control; p Ͻ 0.001)) and UV40 (D 37 ϭ 106 nM versus D 37 ϭ 9.5 nM for vector only control; p Ͻ 0.001) cells to near that of the wild-type parental AA8, confirming previous reports (40,58). However, FANCG(S7A) failed to fully correct the MMC sensitivity of NM3 (D 37 ϭ 40 nM; p Ͻ 0.001) and UV40 (D 37 ϭ 41 nM; p Ͻ 0.001), producing an intermediate phenotype between that of wild-type FANCG and vector-only controls (Fig.…”

“…6B, lanes 3 and 4), which was confirmed as FANCG after membrane stripping and subsequent FANCG immunoblotting. line UV40, which is 20-fold hypersensitive (40,58). Alignment analysis shows that the human FANCG protein shares 70% residue identity to the hamster protein, and this homology extends to the region of serine 7 in the human FANCG (Fig.…”

“…FA-G mutant EUFA143 lymphoblasts were grown in RPMI1640 ϩ 15% fetal bovine serum. The Chinese hamster ovary (CHO) wild-type cell line AA8 and FANCG/XRCC9 mutants NM3 and UV40 were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum as described previously (40).…”

“…Absorbance was measured by A 595 . MMC sensitivity in CHO cell lines was determined by clonal survival assays as described previously (40).…”

Involvement of SLX4 in interstrand cross-link repair is regulated by the Fanconi anemia pathway

“…In higher organisms, ICL processing is orchestrated by the Fanconi anemia (FA) pathway (2,3). Collision of replication forks with ICLs activates the ATR kinase, which in turn licenses the FANCL ubiquitin ligase subunit of the FA core complex (composed of FANCA, B, C, E, F, G, L and M proteins) to modify the FANCD2-FANCI heterodimer (2,(4)(5)(6). The monoubiquitylated FANCD2-FANCI complex is then targeted to chromatin (7,8), where it recruits downstream components of the repairosome, including the structure-specific nuclease FAN1 (9)(10)(11)(12).…”