Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome

Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ-BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an 'undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.     

Fanconi Anemia C Protein Acts at a Switch between Apoptosis and Necrosis in Mitomycin C-Induced Cell Death

Deregulation of apoptosis seems to be a hallmark of the Fanconi anemia (FA) syndrome. In order to further define the role of the FA protein from complementation group C (FAC) in apoptosis, we characterized parameters modified during the mitomycin-C (MMC)-induced apoptotic program. It is shown that despite a higher level of cell death for FA compared to normal lymphoblasts after MMC treatment, FA cells do not display a marked DNA fragmentation. Furthermore, while playing a central role in MMC apoptosis of normal lymphoblasts, the activity of caspase-3-like proteases is altered in FA cells. Interestingly, the disruption of the mitochondrial transmembrane potential (Δψ), an early event that can lead to apoptotic or to necrotic death, is accomplished similarly in FA and in normal cells. Finally, it is shown that the overexpressed FAC protein inhibited the apoptotic steps, with the exception of the decrease of the Δψ. Altogether, our results indicate that the FAC protein acts at a step preceding the activation of the caspases and after the modification of the Δψ, a decision point at which cells can be pushed toward either apoptosis or necrosis and which, consequently, regulates the balance between the two modes of cell death.     

Drosophila FANCM Helicase Prevents Spontaneous Mitotic Crossovers Generated by the MUS81 and SLX1 Nucleases

Several helicases function during repair of double-strand breaks and handling of blocked or stalled replication forks to promote pathways that prevent formation of crossovers. Among these are the Bloom syndrome helicase BLM and the Fanconi anemia group M (FANCM) helicase. To better understand functions of these helicases, we compared phenotypes of Drosophila melanogaster Blm and Fancm mutants. As previously reported for BLM, FANCM has roles in responding to several types of DNA damage in preventing mitotic and meiotic crossovers and in promoting the synthesis-dependent strand annealing pathway for repair of a double-strand gap. In most assays, the phenotype of Fancm mutants is less severe than that of Blm mutants, and the phenotype of Blm Fancm double mutants is more severe than either single mutant, indicating both overlapping and unique functions. It is thought that mitotic crossovers arise when structure-selective nucleases cleave DNA intermediates that would normally be unwound or disassembled by these helicases. When BLM is absent, three nucleases believed to function as Holliday junction resolvases—MUS81-MMS4, MUS312-SLX1, and GEN—become essential. In contrast, no single resolvase is essential in mutants lacking FANCM, although simultaneous loss of GEN and either of the others is lethal in Fancm mutants. Since Fancm mutants can tolerate loss of a single resolvase, we were able to show that spontaneous mitotic crossovers that occur when FANCM is missing are dependent on MUS312 and either MUS81 or SLX1.     

Differential contribution of the Fanconi anemia-related proteins to repair of several types of DNA damage in cultured silkworm cells

The silkworm Fanconi anemia (FA) pathway is required for normal cellular resistance to mitomycin C (MMC) in silkworms, but little is known about the requirement for repair of other types of DNA damage. Here we report that silkworm cells deficient for FA proteins FancD2 and FancM exhibit normal sensitivities to hydroxyurea (HU) and camptothecin (CPT), although FancM-dependent FancD2 monoubiquitination is induced upon these treatments. Similar results were observed in cells depleted for Rmi1 and Mhf1, which interact with the FancM protein. We also found that Rad51-knockdown cells exhibited normal sensitivity to HU despite induction of double-strand breaks by HU treatment.     

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.     

Male Fertility Defect Associated with Disrupted BRCA1-PALB2 Interaction in Mice

PALB2 links BRCA1 and BRCA2 in homologous recombinational repair of DNA double strand breaks (DSBs). Mono-allelic mutations in PALB2 increase the risk of breast, pancreatic, and other cancers, and biallelic mutations cause Fanconi anemia (FA). Like Brca1 and Brca2, systemic knock-out of Palb2 in mice results in embryonic lethality. In this study, we generated a hypomorphic Palb2 allele expressing a mutant PALB2 protein unable to bind BRCA1. Consistent with an FA-like phenotype, cells from the mutant mice showed hypersensitivity and chromosomal breakage when treated with mitomycin C, a DNA interstrand crosslinker. Moreover, mutant males showed reduced fertility due to impaired meiosis and increased apoptosis in germ cells. Interestingly, mutant meiocytes showed a significant defect in sex chromosome synapsis, which likely contributed to the germ cell loss and fertility defect. Our results underscore the in vivo importance of the PALB2-BRCA1 complex formation in DSB repair and male meiosis.     

What is wrong with Fanconi anemia cells?

Figuring out what is wrong in Fanconi anemia (FA) patient cells is critical to understanding the contributions of the FA pathway to DNA repair and tumor suppression. Although FA patients exhibit a wide range of disease manifestation as well as severity (asymptomatic to congenital abnormalities, bone marrow failure, and cancer), cells from FA patients share underlying defects in their ability to process DNA lesions that interfere with DNA replication. In particular, FA cells are very sensitive to agents that induce DNA interstrand crosslinks (ICLs). The cause of this pronounced ICL sensitivity is not fully understood, but has been linked to the aberrant activation of DNA damage repair proteins, checkpoints and pathways. Thus, regulation of these responses through coordination of repair processing at stalled replication forks is an essential function of the FA pathway. Here, we briefly summarize some of the aberrant DNA damage responses contributing to defects in FA cells, and detail the newly-identified relationship between FA and the mismatch repair protein, MSH2. Understanding the contribution of MSH2 and/or other proteins to the replication problem in FA cells will be key to assessing therapeutic options to improve the health of FA patients. Moreover, loss of these factors, if linked to improved replication, could be a key event in the progression of FA cells to cancer cells. Likewise, loss of these factors could synergize to enhance tumorigenesis or confer chemoresistance in tumors defective in FA-BRCA pathway proteins and provide a basis for biomarkers for disease progression and response.     

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.     

DNA2 and EXO1 in replication-coupled,homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.     

The Fanconi anemia pathway and DNA interstrand cross-link repair

Fanconi anemia (FA) is an autosomal or X-linked recessive disorder characterized by chromosomal instability, bone marrow failure, cancer susceptibility, and a profound sensitivity to agents that produce DNA interstrand cross-link (ICL). To date, 15 genes have been identified that, when mutated, result in FA or an FA-like syndrome. It is believed that cellular resistance to DNA interstrand cross-linking agents requires all 15 FA or FA-like proteins. Here, we review our current understanding of how these FA proteins participate in ICL repair and discuss the molecular mechanisms that regulate the FA pathway to maintain genome stability.