In this study, we provide genetic evidence for a contributory role of FANCG
in HR as measured by three different assays. First, HR-mediated repair of I-Sce
I-induced chromosomal DSBs was reduced in fang
mutant cells. Second, the overall efficiency of gene targeting was mildly decreased at three of four loci examined. Third, the levels of chromosome aberrations were elevated in X-irradiated cells in late S to G2
phase compared with cells irradiated in other phases. Given a primary role of HR in repairing IR-induced DSBs in late S and G2
phases in DT40 cells, compared with the secondary role of nonhomologous end joining (52
), these findings are consistent with a role of FANCG in HR-mediated repair.
However, other HR-related processes examined were unaffected in the fancg cells. Specifically, the frequency of spontaneous or MMC-induced SCE appears to be normal. SCEs may be considered a surrogate marker of recombination efficiency, in contrast to chromosomal aberrations, which reflect biologically relevant HR repair. Moreover, the IR sensitivity of asynchronous fancg cells was normal, which differs from the case for rad51 paralog mutants. Thus, FANCG in DT40 cells seems to be important for optimal activity of only selected processes that depend on HR.
It has been shown that the FA multiprotein complex's assembly and nuclear localization is impaired in human fancg
). However, at present it is not clear whether defective complex localization underlies the entire phenotype of fancg
cells. For example, human FANCG has been reported to interact directly with cytoplasmic CYP2E1 and appears to down-regulate the level of CYP2E1, a potential generator of reactive oxidative species (ROS) (14
The FANCG protein likely has a function different from that of BRCA2 or RAD51 paralogs in HR for several reasons. First, we found no change in Rad51 focus formation in fancg
cells, in keeping with one recent report (18
) but not with another (9
). Second, overexpression of human Rad51 did not increase cisplatin resistance of fancg
mutant cells, whereas the defects in Rad51 paralog mutants were compensated for by elevating Rad51 protein (51
). (The effect of Rad51 overexpression in brca2
DT40 cells remains to be clarified.) We suggest that FANCG may function in parallel with BRCA2 by leading to common phenotypic consequences. It will be interesting to test this hypothesis by making mutant cells deficient in both BRCA2
Given the possibility that ATM regulates BRCA2 (58
), this relationship may be relevant to the idea that FANCD2, which is also a phosphorylation target of ATM (54
), may represent a convergence of two separate pathways, e.g., ATM signaling and the function of the FA nuclear complex.
So far, with the exception of BRCA2, no direct interactions between FA proteins and repair enzymes such as Rad51 and Mre11 have been identified. The defects in fancg
cells are consistently milder than those observed in other HR-defective mutants such as the Rad51 paralogs (45
). These facts seem to be consistent with the view that FANCG plays an indirect, facilitating role in HR. For example, the FA proteins may function in part through chromatin remodeling (41
). FANCA was reported to interact with Brg1 (37
), a component of the human SWI/SNF complex, and FANCC interacts with the transcriptional repressor protein FAZF (22
). It is possible that defective chromatin remodeling could influence cellular HR capacity (such as gene targeting efficiency) by modulating transcription, or temporal expression, of genes directly involved in HR.
Accumulating evidence supports a role for the FA proteins in determining the level of cytoplasmic ROS (reviewed in reference 1
). Specifically, ROS may be elevated in FA group G human lymphoblasts, since they are reported to have elevated oxidative DNA damage, which is reversed in FANCG
-complemented cells (14
). Importantly, DNA damage by ROS appears to be a major cause of spontaneous chromosome breaks (28
). Thus, the extensive chromosome aberrations often seen in FA cells (but not fancg
DT40 cells) may be due to increased DNA damage by excessive generation of ROS along with compromised damage response processes linked to HR.