Preventing over-resection by DNA2 helicase/nuclease suppresses repair defects in Fanconi anemia cells

FANCD2 is required for the repair of DNA damage by the FA (Fanconi anemia) pathway, and, consequently, FANCD2-deficient cells are sensitive to compounds such as cisplatin and formaldehyde that induce DNA:DNA and DNA:protein crosslinks, respectively. The DNA2 helicase/nuclease is required for RNA/DNA flap removal from Okazaki fragments during DNA replication and for the resection of DSBs (double-strand breaks) during HDR (homology-directed repair) of replication stress-induced damage. A knockdown of DNA2 renders normal cells as sensitive to cisplatin (in the absence of EXO1) and to formaldehyde (even in the presence of EXO1) as FANCD2^−/− cells. Surprisingly, however, the depletion of DNA2 in FANCD2-deficient cells rescues the sensitivity of FANCD2^−/− cells to cisplatin and formaldehyde. We previously showed that the resection activity of DNA2 acts downstream of FANCD2 to insure HDR of the DSBs arising when replication forks encounter ICL (interstrand crosslink) damage. The suppression of FANCD2^−/− by DNA2 knockdowns suggests that DNA2 and FANCD2 also have antagonistic roles: in the absence of FANCD2, DNA2 somehow corrupts repair. To demonstrate that DNA2 is deleterious to crosslink repair, we used psoralen-induced ICL damage to trigger the repair of a site-specific crosslink in a GFP reporter and observed that “over-resection” can account for reduced repair. Our work demonstrates that excessive resection can lead to genome instability and shows that strict regulatory processes have evolved to inhibit resection nucleases. The suppression of FANCD2^−/− phenotypes by DNA2 depletion may have implications for FA therapies and for the use of ICL-inducing agents in chemotherapy.     

Effect of caffeine in Fanconi anemia

Summary In Fanconi anemia (FA) cells the duration of the G₂ phase of the cell cycle prolonged. Such a slowing of the G₂ phase can be induced in normal cells by irradiation with rays during S phase, which also further increases the duration of G₂ in FA cells. The addition of caffeine during the last 7h of culture shortens the G₂ phase in both nonirradiated and irradiated FA cells. In nonirradiated normal cells it may have no effect or may increase G₂ phase duration, but in irradiated normal reduces the slowing of G₂ induced by the radiation. This suggests that FA cells recognize and repair preexisting DNA lesions during G₂ phase and that caffeine inhibits this process. The principal anomaly in FA may be a deficient repair during S phase, as manifest in the prolonged postreplication repair period during G₂ phase required to repair the larger number of lesions passing through S phase.     

Fanconi anemia proteins in telomere maintenance

Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Telomeres ensure genome stability by preventing chromosome termini from being recognized as DNA damage. Telomere length homeostasis is inevitable for telomere maintenance because critical shortening or over-lengthening of telomeres may lead to DNA damage response or delay in DNA replication, and hence genome instability. Due to their repetitive DNA sequence, unique architecture, bound shelterin proteins, and high propensity to form alternate/secondary DNA structures, telomeres are like common fragile sites and pose an inherent challenge to the progression of DNA replication, repair, and recombination apparatus. It is conceivable that longer the telomeres are, greater is the severity of such challenges. Recent studies have linked excessively long telomeres with increased tumorigenesis. Here we discuss telomere abnormalities in a rare recessive chromosomal instability disorder called Fanconi Anemia and the role of the Fanconi Anemia pathway in telomere biology. Reports suggest that Fanconi Anemia proteins play a role in maintaining long telomeres, including processing telomeric joint molecule intermediates. We speculate that ablation of the Fanconi Anemia pathway would lead to inadequate aberrant structural barrier resolution at excessively long telomeres, thereby causing replicative burden on the cell.     

Mutation analysis of the Fanconi anemia gene FACC.

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive disorder characterized by a unique hypersensitivity of cells to DNA cross-linking agents; a gene for complementation group C (FACC) has recently been cloned. We have amplified FACC exons with their flanking intron sequences from genomic DNA from 174 racially and ethnically diverse families in the International Fanconi Anemia Registry and have screened for mutations by using SSCP analysis. We identified eight different variants in 32 families; three were detected in exon 1, one in exon 4, one in intron 4, two in exon 6, and one in exon 14. Two of the eight variants, in seven families, did not segregate with the disease allele in multiplex families, suggesting that these variants represented benign polymorphisms. Disease-associated mutations in FACC were detected in a total of 25 (14.4%) of 174 families screened. The most frequent mutations were IVS4 + 4 A-->T (intron 4; 12 families) and 322delG (exon 1; 9 families). Other, less common mutations include Q13X in exon 1, R185X and D195V in exon 6, and L554P in exon 14. The polymorphisms were S26F in exon 1 and G139E in exon 4. All patients in our study with 322delG, Q13X, R185X, and D195V are of northern or eastern European or southern Italian ancestry, and 18 of 19 have a mild form of the disease, while the 2 patients with L554P, both from the same family, have a severe phenotype. All 19 patients with IVS4 + 4 A-->T have Jewish ancestry and have a severe phenotype.     

The Fanconi anemia signalosome anchor

FAAP24, a new XPF endonuclease family member identified by in a recent issue of Molecular Cell, heterodimerizes with FANCM, binds unwound DNA, and reveals how the Fanconi anemia core complex concentrates DNA repair proteins at stalled replication forks.     

Disease model: Fanconi anemia

Fanconi anemia (FA) is a chromosomal instability syndrome characterized by the presence of pancytopenia, congenital malformations and cancer predisposition. Six genes associated with this disorder have been cloned, and mice with targeted disruptions of several of the FA genes have been generated. These mouse models display the characteristic FA feature of cellular hypersensitivity to DNA cross-linking agents. Although they do not develop hematological or developmental abnormalities spontaneously, they mimic FA patients in their reduced fertility. Studies using these animal models provide valuable insights into the involvement of apoptotic pathways in FA, and help characterize the defects in FA hematopoietic cells. In addition, mouse models are also useful for testing treatments for FA.     

Antioxidant status of Fanconi anemia fibroblasts

Summary Several observations in the recent literature have indicated that Fanconi anemia (FA) cells may be primarily deficient in the detoxification of activated oxygen species. To evaluate the antioxidant status of FA fibroblasts, we measured Mn-containing superoxide dismutase (Mn-SOD), CuZn-containing superoxide dismutase (CuZn-SOD), catalase, and glutathione peroxidase activities, as well as cellular glutathione contents and total nonenzymatic antioxidant potential in Fa and control fibroblasts at multiple time point during a single passage. All parameters exhibited a characteristic pattern of changes during a period of 19 days following trypsinization. Unlike FA erythrocytes, which are known to be deficient in CuZn-SOD, FA fibroblasts exhibited normal CuZn-SOD activities. Also, the nonenzymatic antioxidant potential as well as glutathione levels were similar in FA and control fibroblasts. However, Mn-SOD, catalase, and glutathione peroxidase activities were consistently higher in FA fibroblasts. We hypothesize that the elevation of these enzyme activities might reflect a cellular prooxidant state in FA resulting from an increased formation of endogenous oxidizing molecular species that trigger enhanced synthesis of certain enzymatic antioxidant defenses.     

Evidence for at least eight Fanconi anemia genes.

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.     

Growth of cultured cells from patients with Fanconi anemia.

Fibroblast cultures derived from skin biopsies of patients with Fanconi anemia had doubling times (mean of five lines: 30.3 +/- 0.2 hours) significantly longer than randomly selected normal controls (mean of nine lines: 22.9 +/- 0.4 hours). Control cultures grew more slowly in the enriched media RPMI 1640 and McCoy's 5A than in MEM; while a culture from a patient with Fanconi anemia grew more slowly only in McCoy's 5A. Differences in growth characteristics between Fanconi anemia and normal cell cultures may be useful in analyzing the metabolic error determined by the Fanconi anemia gene.     

Fanconi anemia proteins stabilize replication forks

Fanconi anemia (FA) is a recessive genetic disorder characterized by hypersensitivity to crosslinking agents that has been attributed to defects in DNA repair and/or replication. FANCD2 and the FA core complex bind to chromatin during DNA replication; however, the role of FA proteins during replication is unknown. Using Xenopus cell-free extracts, we show that FANCL depletion results in defective DNA replication restart following treatment with camptothecin, a drug that results in DSBs during DNA replication. This defect is more pronounced following treatment with mitomycin C, presumably because of an additional role of the FA pathway in DNA crosslink repair. Moreover, we show that chromatin binding of FA core complex proteins during DNA replication follows origin assembly and origin firing and is dependent on the binding of RPA to ssDNA while FANCD2 additionally requires ATR, consistent with FA proteins acting at replication forks. Together, our data suggest that FA proteins play a role in replication restart at collapsed replication forks.     

Ubiquitylation and the Fanconi anemia pathway

The Fanconi anemia (FA) pathway maintains genome stability through co-ordination of DNA repair of interstrand crosslinks (ICLs). Disruption of the FA pathway yields hypersensitivity to interstrand crosslinking agents, bone marrow failure and cancer predisposition. Early steps in DNA damage dependent activation of the pathway are governed by monoubiquitylation of FANCD2 and FANCI by the intrinsic FA E3 ubiquitin ligase, FANCL. Downstream FA pathway components and associated factors such as FAN1 and SLX4 exhibit ubiquitin-binding motifs that are important for their DNA repair function, underscoring the importance of ubiquitylation in FA pathway mediated repair. Importantly, ubiquitylation provides the foundations for cross-talk between repair pathways, which in concert with the FA pathway, resolve interstrand crosslink damage and maintain genomic stability.     

Fanconi anemia and DNA replication repair

There has been a recent profusion of reviews on Fanconi anemia (FA), which will give readers a comprehensive outline of the field R.D. Kennedy, A.D. D'Andrea, The Fanconi anemia/BRCA pathway: new faces in the crowd, Genes Dev. 19 (2005) 2925-2940; L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked to DNA repair, Cell 123 (2005) 1191-1198; H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anaemia, Nat. Rev. Genet. 2 (2001) 446-457. Here, we will focus on key areas that place the FA proteins in the context of DNA repair during replication. In addition, where possible we will put forward propositions that in our opinion need addressing, and where possible provide models that can be tested.     

The cell cycle of lymphocytes in Fanconi anemia

Summary BrdU-incorporation techniques were used to study the cell cycle in 18 cases of Fanconi's anemia (FA).By comparison with controls, a significant slowing of the cell cycle of lymphocytes in vitro was observed in all FA patients, and possibly in FA heterozygotes, although to a lesser degree. It is probable that the demonstration of the slowing is dependent on the culture conditions. No slowing was observed in other patients affected by at least one of the symptoms of FA. The slow cell cycle of FA cells is mostly due to a very long G₂-phase. A relationship between slow cell cycle and chromatid anomalies exists, the slower cells being significantly more frequently carriers of radial figures than the faster cells, in the same patient.     

Fanconi anemia mutation causes cellular susceptibility to ambient oxygen.

The gene defect causing the Fanconi anemia (FA) phenotype appears to be expressed at the cellular level, since FA fibroblasts show a protracted course of explant outgrowth, a diminished in vitro life span, and very poor cloning. We show that exposure of FA fibroblasts to hypoxic (5% v/v oxygen) culture conditions restores their growth in vitro to near normal. Exposure to elevated oxygen tension (35% v/v) causes accumulations of FA cells in the S and G2/M phases of the cell cycle that are in significant excess of those seen in heterozygote and control strains. In the absence of evidence for defective cytoplasmatic radical scavenging systems, these observations suggest increased nuclear susceptibility to ambient oxygen as cause of the FA cellular phenotype.