Abdullah Mahmood Ali

BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the Bloom helicase-double Holliday junction dissolvasome.

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIalpha and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo IIIalpha-BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIalpha and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.

MHF1-MHF2, a histone-fold-containing protein complex, participates in the Fanconi anemia pathway via FANCM.

FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.

FAAP100 is essential for activation of the Fanconi anemia-associated DNA damage response pathway

The Fanconi anemia (FA) core complex plays a central role in the DNA damage response network involving breast cancer susceptibility gene products, BRCA1 and BRCA2. The complex consists of eight FA proteins, including a ubiquitin ligase (FANCL) and a DNA translocase (FANCM), and is essential for monoubiquitination of FANCD2 in response to DNA damage. Here, we report a novel component of this complex, termed FAAP100, which is essential for the stability of the core complex and directly interacts with FANCB and FANCL to form a stable subcomplex. Formation of this subcomplex protects each component from proteolytic degradation and also allows their coregulation by FANCA and FANCM during nuclear localization. Using siRNA depletion and gene knockout techniques, we show that FAAP100‐deficient cells display hallmark features of FA cells, including defective FANCD2 monoubiquitination, hypersensitivity to DNA crosslinking agents, and genomic instability. Our study identifies FAAP100 as a new critical component of the FA‐BRCA DNA damage response network.

FAAP20: a novel ubiquitin-binding FA nuclear core-complex protein required for functional integrity of the FA-BRCA DNA repair pathway

Fanconi anemia (FA) nuclear core complex is a multiprotein complex required for the functional integrity of the FA-BRCA pathway regulating DNA repair. This pathway is inactivated in FA, a devastating genetic disease, which leads to hematologic defects and cancer in patients. Here we report the isolation and characterization of a novel 20-kDa FANCA-associated protein (FAAP20). We show that FAAP20 is an integral component of the FA nuclear core complex. We identify a region on FANCA that physically interacts with FAAP20, and show that FANCA regulates stability of this protein. FAAP20 contains a conserved ubiquitin-binding zinc-finger domain (UBZ), and binds K-63-linked ubiquitin chains in vitro. The FAAP20-UBZ domain is not required for interaction with FANCA, but is required for DNA-damage-induced chromatin loading of FANCA and the functional integrity of the FA pathway. These findings reveal critical roles for FAAP20 in the FA-BRCA pathway of DNA damage repair and genome maintenance.

ATR-Dependent Phosphorylation of FANCM at Serine 1045 Is Essential for FANCM Functions

Fanconi anemia (FA) is a genome instability syndrome that has been associated with both cancer predisposition and bone marrow failure. FA proteins are involved in cellular response to replication stress in which they coordinate DNA repair with DNA replication and cell-cycle progression. One regulator of the replication stress response is the ATP-dependent DNA translocase FANCM, which we have shown to be hyperphosphorylated in response to various genotoxic agents. However, the significance of this phosphorylation remained unclear. Here, we show that genotoxic stress-induced FANCM phosphorylation is ATR-dependent and that this modification is highly significant for the cellular response to replication stress. We identified serine (S1045) residue of FANCM that is phosphorylated in response to genotoxic stress and this effect is ATR-dependent. We show that S1045 is required for FANCM functions including its role in FA pathway integrity, recruiting FANCM to the site of interstrand cross links, preventing the cells from entering mitosis prematurely, and efficient activation of the CHK1 and G2-M checkpoints. Overall, our data suggest that an ATR-FANCM feedback loop is present in the FA and replication stress response pathways and that it is required for both efficient ATR/CHK1 checkpoint activation and FANCM function.

Ectopic HOXB4 overcomes the inhibitory effect of tumor necrosis factor-α on Fanconi anemia hematopoietic stem and progenitor cells

Ectopic delivery of HOXB4 elicits the expansion of engrafting hematopoietic stem cells (HSCs). We hypothesized that inhibition of tumor necrosis factor-alpha (TNF-alpha) signaling may be central to the self-renewal signature of HOXB4. Because HSCs derived from Fanconi anemia (FA) knockout mice are hypersensitive to TNF-alpha, we studied Fancc(-/-) HSCs to determine the physiologic effects of HOXB4 on TNF-alpha sensitivity and the relationship of these effects to the engraftment defect of FA HSCs. Overexpression of HOXB4 reversed the in vitro hypersensitivity to TNF-alpha of Fancc(-/-) HSCs and progenitors (P) and partially rescued the engraftment defect of these cells. Coexpression of HOXB4 and the correcting FA-C protein resulted in full correction compared with wild-type (WT) HSCs. Ectopic expression of HOXB4 resulted in a reduction in both apoptosis and reactive oxygen species in Fancc(-/-) but not WT HSC/P. HOXB4 overexpression was also associated with a significant reduction in surface expression of TNF-alpha receptors on Fancc(-/-) HSC/P. Finally, enhanced engraftment was seen even when HOXB4 was expressed in a time-limited fashion during in vivo reconstitution. Thus, the HOXB4 engraftment signature may be related to its effects on TNF-alpha signaling, and this pathway may be a molecular target for timed pharmacologic manipulation of HSC during reconstitution.

FANCM-FAAP24 and FANCJ: FA proteins that metabolize DNA.

Fanconi anemia (FA) is a rare autosomal recessive or X-linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA-crosslinking agents. Eight FA proteins (FANCA, -B, -C, -E, -F, -G, -L and -M) and three non-FA proteins (FAAP100, FAAP24 and HES1) form the FA nuclear core complex that is required for monoubiquitination of the FANCD2-FANCI dimer upon DNA damage. The other three FA proteins, FANCD1/BRCA2, FANCJ/BACH1/BRIP1 and FANCN/PALB2, act in parallel or downstream of the FANCD2-FANCI dimer. Despite the isolation and characterization of several FA proteins, the mechanism by which these proteins protect cells from DNA interstrand crosslinking agents has been unclear. This is because a majority of the FA proteins lack any recognizable functional domains that can provide insight into their function. The recently discovered FANCM (Hef) and FANCJ (BRIP1/BACH1) proteins contain helicase domains, providing potential insight into the role of FA proteins in DNA repair. FANCM with its partner, FAAP24, and FANCJ bind and metabolize a variety of DNA substrates. In this review, we focus on the discovery, structure, and function of the FANCM-FAAP24 and FANCJ proteins.

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.

Identification and Characterization of Mutations in FANCL Gene: a Second Case of Fanconi Anemia Belonging to FA-L Complementation Group

Fanconi anemia (FA) is a rare autosomal recessive or X‐linked disorder characterized by aplastic anemia, cancer susceptibility and cellular sensitivity to DNA crosslinking agents. Eight FA proteins (FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL and FANCM) and three non‐FA proteins (FAAP100, FAAP24 and HES1) form an FA nuclear core complex, which is required for monoubiquitination of the FANCD2‐FANCI dimer upon DNA damage. FANCL possesses a PHD/RING‐finger domain and is a putative E3 ubiquitin ligase subunit of the core complex. In this study, we report an FA patient with an unusual presentation belonging to the FA‐L complementation group. The patient lacks an obvious FA phenotype except for the presence of a café‐au‐lait spot, mild hypocellularity and a family history of leukemia. The molecular diagnosis and identification of the FA subgroup was achieved by FA complementation assay. We identified bi‐allelic novel mutations in the FANCL gene and functionally characterized them. To the best of our knowledge, this is the second reported case belonging to the FA‐L complementation group.

Monopolar Spindle 1 (MPS1) Protein-dependent Phosphorylation of RecQ-mediated Genome Instability Protein 2 (RMI2) at Serine 112 Is Essential for BLM-Topo III α-RMI1-RMI2 (BTR) Protein Complex Function upon Spindle Assembly Checkpoint (SAC) Activation during Mitosis

Background: The BTR complex is required for genomic stability and is posttranslationally modified in cells arrested at the mitotic spindle assembly checkpoint (SAC). Results: We identify serine 112 (Ser-112) as an MPS1-dependent phosphorylation site on RMI2. Conclusion: MPS1-dependent phosphorylation of RMI2 regulates the chromosomal maintenance upon SAC activation during mitosis. Significance: Phosphorylation of RMI2 regulates SAC checkpoint signaling and maintains genomic stability. Genomic instability and a predisposition to cancer are hallmarks of Bloom syndrome, an autosomal recessive disease arising from mutations in the BLM gene. BLM is a RecQ helicase component of the BLM-Topo III α-RMI1-RMI2 (BTR) complex, which maintains chromosome stability at the spindle assembly checkpoint (SAC). Other members of the BTR complex include Topo IIIa, RMI1, and RMI2. All members of the BTR complex are essential for maintaining the stable genome. Interestingly, the BTR complex is posttranslationally modified upon SAC activation during mitosis, but its significance remains unknown. In this study, we show that two proteins that interact with BLM, RMI1 and RMI2, are phosphorylated upon SAC activation, and, like BLM, RMI1, and RMI2, are phosphorylated in an MPS1-dependent manner. An S112A mutant of RMI2 localized normally in cells and was found in SAC-induced coimmunoprecipitations of the BTR complex. However, in RMI2-depleted cells, an S112A mutant disrupted the mitotic arrest upon SAC activation. The failure of cells to maintain mitotic arrest, due to lack of phosphorylation at Ser-112, results in high genomic instability characterized by micronuclei, multiple nuclei, and a wide distribution of aberrantly segregating chromosomes. We found that the S112A mutant of RMI2 showed defects in redistribution between the nucleoplasm and nuclear matrix. The phosphorylation at Ser-112 of RMI2 is independent of BLM and is not required for the stability of the BTR complex, BLM focus formation, and chromatin targeting in response to replication stress. Overall, this study suggests that the phosphorylation of the BTR complex is essential to maintain a stable genome.