Human MutS and FANCM complexes function as redundant DNA damage sensors in the Fanconi Anemia pathway

The Fanconi Anemia (FA) pathway encodes a DNA damage response activated by DNA damage-stalled replication forks. Current evidence suggests that the FA pathway initiates with DNA damage recognition by the FANCM complex (FANCM/FAAP24/MHF). However, genetic inactivation of FANCM in mouse and DT40 cells causes only a partial defect in the FA pathway activation, suggesting the existence of redundant DNA damage sensors. Here we show that the MutS homologs function in this capacity. A RNAi screen revealed that MSH2 silencing caused defective FA pathway activation, as assessed by damage-induced FANCD2 mono-ubiquitination. A similar FA pathway defect was observed with MSH3 or MSH6 silencing. MSH2 depletion caused cellular phenotypes associated with defective FA pathway, including mitomycin C hypersensitivity and chromosomal instability. Further, silencing of FANCM in MSH2 deficient HEC59 cells caused a more severe FA defect relative to comparable silencing in MSH2 complemented HEC59 + Chr2 cells, suggesting redundant functions between MSH2 and FANCM. Consistent with this hypothesis, depletion of MSH2 resulted in defective chromatin localization of the FA core complex upon DNA damage. Further, MSH2 was co-purified and co-immunoprecipitated with FA core complex components. Taken together, our results suggest that human MutS homologs and FANCM complexes function as redundant DNA damage sensors of the FA pathway.     

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G₂/M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.     

范可尼贫血与泛素化

Fanconi贫血是一种罕见的隐性遗传性疾病,临床常以先天性畸形、进行性骨髓衰竭和遗传性肿瘤倾向为主要表现而确诊。FA病人细胞对DNA交联剂如丝裂霉素C (MMC)高度敏感。目前已经发现至少12种FA基因的缺失或突变能够引起FA表型的出现,其中10种相应的编码蛋白形成FA复合物共同参与FA/BRCA2 DNA损伤修复途径—FA途径。FA核心复合物蛋白FANCL具有泛素连接酶活性,在结合酶UBE2T共同作用下,催化下游蛋白FANCD2单泛化,泛素化FANCD2与BRCA2形成新的复合物,修复DNA损伤。去泛素化酶USP1在DNA修复完毕后移除FANCD2的单体泛素,使因损伤修复而阻滞的细胞周期继续进行。机体很可能在不同信号通路对FANCD2泛素化/去泛素化的精细调节下,调控FA途径参与不同的DNA修复过程。     

Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.     

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1-/- mice

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.     

Impairment of homologous recombination control in a Fanconi anemia-like Chinese hamster cell mutant

DNA interstrand cross-links (ICL)-inducing agents such as cisplatin, mitomycin C (MMC) and nitrogen mustards are widely used as potent antitumor drugs. Although ICL repair mechanism is not yet well characterized in mammalian cells, this pathway is thought to involve a sequential action of nucleotide excision repair (NER) and homologous recombination (HR). The importance of unraveling ICL repair pathways is highlighted by the hypersensitivity to ICL-inducing agents in cells of patients with the genetic disease Fanconi anemia (FA) and in cells mutated in the Breast Cancer susceptibility genes BRCA1 and BRCA2. To better characterize the involvement of HR in the sensitivity to ICL-inducing agents, we examined spontaneous and ICL-induced HR in rodent FA-like V-H4 cells. In this report, we show that MMC-hypersensitive V-H4 cells exhibit an increased spontaneous homology-directed repair (HDR) activity compared to the resistant V79 parental cells. Elevated HDR activity results mainly in increased conservative Rad51-dependent recombination, without affecting non-conservative single-strand annealing process (SSA). We also show that HDR activity is enhanced following MMC treatment in parental cells, but not in rodent FA-like V-H4 cells. Moreover, our data indicate that Rad51 foci formation is significantly delayed in these FA-like cells in response to crosslinking agent. These findings provide evidence for an impairment of HR control in V-H4 cells and emphasize the involvement of the FA pathway in HR-mediated repair.     

Mgm101: A double-duty Rad52-like protein

Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues (Mph1, Chl1 and Slx4), this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutSα (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in rad52 defective cells. We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs (such as at stalled replication forks, broken chromosomes and natural chromosome ends), stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.     

Crosstalk between the nucleotide excision repair and Fanconi anemia/BRCA pathways

Cells have evolved multiple distinct DNA repair pathways to efficiently correct a variety of genotoxic lesions, and decades of study have led to an improved understanding of the mechanisms and regulation of these individual pathways. However, there is now an increasing appreciation that extensive crosstalk exists among DNA repair pathways and that this crosstalk serves to increase the efficiency and diversity of response to damage. The Fanconi anemia (FA)/BRCA and nucleotide excision repair (NER) pathways have been shown to share common factors, and often work in concert to repair damage. Genomic studies are now revealing that many tumors harbor somatic mutations in FA/BRCA or NER genes, which may provide a growth advantage, but which could also be exploited therapeutically.