SUMMARY FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here we show that FANCM forms a conserved DNA remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA remodeling complex that protects replication forks from yeast to human.

Fanconi anemia (FA) is a genetically heterogeneous cancer-prone disorder associated with chromosomal instability and cellular hypersensitivity to DNA crosslinking agents. The FA pathway is suspected to play a crucial role in the cellular response to DNA replication stress. At a molecular level, however, the function of most of the FA proteins is unknown. FANCM displays DNA-dependent ATPase activity and promotes the dissociation of DNA triplexes, but the physiological significance of this activity remains elusive. Here we show that purified FANCM binds to Holliday junctions and replication forks with high specificity and promotes migration of their junction point in an ATPase-dependent manner. Furthermore, we provide evidence that FANCM can dissociate large recombination intermediates, via branch migration of Holliday junctions through 2.6 kb of DNA. Our data suggest a direct role for FANCM in DNA processing, consistent with the current view that FA proteins coordinate DNA repair at stalled replication forks.

Proteins disabled in Fanconi anemia (FA) are necessary for the maintenance of genome stability during cell proliferation. Upon replication stress signaling by ATR, the FA core complex monoubiquitinates FANCD2 and FANCI in order to activate DNA repair. Here, we identified FANCD2 and FANCI in a proteomic screen of replisome-associated factors bound to nascent DNA in response to replication arrest. We found that FANCD2 can interact directly with minichromosome maintenance (MCM) proteins. ATR signaling promoted the transient association of endogenous FANCD2 with the MCM2-MCM7 replicative helicase independently of FANCD2 monoubiquitination. FANCD2 was necessary for human primary cells to restrain DNA synthesis in the presence of a reduced pool of nucleotides and prevented the accumulation of single-stranded DNA, the induction of p21, and the entry of cells into senescence. These data reveal that FANCD2 is an effector of ATR signaling implicated in a general replisome surveillance mechanism that is necessary for sustaining cell proliferation and attenuating carcinogenesis.

The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA-or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.signal transduction | T-cell receptor T he antigen receptor-driven activation of the nuclear factorkappa B (NF-κB) signaling pathway has recently gained considerable interest because genetic deficiencies in this pathway are linked to immune deficiencies, whereas aberrant constitutive NF-κB activation is associated with the development of autoimmune disease and neoplastic disorders (1-4).The NF-κB family of transcription factors comprises five transcription factors that share a Rel homology domain (RHD) required for DNA binding and homo-or heterodimerization (1, 5). The transcriptionally active form of NF-κB is a heterodimer containing a member with an RHD (p50 or p52) and one with an RHD and an additional transcription activation domain (RelA, RelB, or c-Rel). NF-κB family members are present in the cytoplasm in an inactive form that can be mobilized by either the classical (canonical) or alternative (noncanonical) pathway. The classical pathway is thought to control the activation of p50-RelA and p50-c-Rel complexes by stimulus-dependent degradation of inhibitor of κB (IκB) proteins that bind these complexes and prevent their nuclear translocation. The alternative pathway, on the other hand, controls the generation of transcriptionally active p52-RelB complexes through stimulationinduced processing of the p52 precursor p100 (5).T-cell receptor (TCR)-induced NF-κB activation has been shown to rely on the activation of both RelA-and c-Rel-containing NF-κB complexes that occur in a timely staggered manner (6). However, the exact mechanism controlling persistent RelA-p50 and c-Rel-p50 activation in T cells remains not well characterized.Biochemical and genetic studies have identified an essential role for proteins of the Carma1/Bcl-10/Malt1 (CBM) signaling module in TCR-induced NF-κB activation (2, 7-9). The current model of CBM-dependent NF-κB activation suggests that antigen triggering leads to the phosphorylation of Carma1 by PKC family and probably additional kinases, inducing a conformational change in Carma1 that allows it to recruit preformed Bcl-10/Malt1 complexes (7, 10). Malt1 is thought to control the activation of the IKK complex by binding to the ubiquitin ligase T...

Fanconi anemia (FA) is a genetically heterogeneous chromosome instability syndrome associated with congenital abnormalities, bone marrow failure, and cancer predisposition. Eight FA proteins form a nuclear core complex, which promotes tolerance of DNA lesions in S phase, but the underlying mechanisms are still elusive. We reported recently that the FA core complex protein FANCM can translocate Holliday junctions. Here we show that FANCM promotes reversal of model replication forks via concerted displacement and annealing of the nascent and parental DNA strands. Fork reversal by FANCM also occurs when the lagging strand template is partially single-stranded and bound by RPA. The combined fork reversal and branch migration activities of FANCM lead to extensive regression of model replication forks. These observations provide evidence that FANCM can remodel replication fork structures and suggest a mechanism by which FANCM could promote DNA damage tolerance in S phase.fanconi anemia ͉ replication fork A variety of structural and chemical alterations in DNA can hinder the progression of replication forks and precipitate the formation of gross chromosomal rearrangements. These hurdles impose distinct structural constraints in the template DNA, which elicit the action of diverse lesion bypass or lesion tolerance pathways (1, 2). Covalent links between complementary DNA strands constitute a unique challenge to replicating cells, because they preclude strand separation and, hence, completely block fork progression. In mammalian cells, the repair of DNA interstrand cross-links (ICLs) is thought to take place during S phase (3). The exact mechanism of repair is unknown, but it seems to involve the interplay of different pathways, with the homologous recombination machinery, translesion DNA polymerases, and the Fanconi anemia (FA) pathway all being required for ICL tolerance (4).FA is a genetically heterogeneous inherited disorder, which combines congenital abnormalities, bone marrow failure, and a marked cancer predisposition (5-8). FA cells are prone to spontaneous and damage-induced chromosomal aberrations and are notoriously hypersensitive to DNA interstrand cross-linking agents. FA proteins can be classified into three groups (8). Group I includes FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM. These eight FA proteins form a nuclear core complex (9-11) whose integrity is required for the conjugation of a ubiquitin moiety to the group II proteins, FANCI and FANCD2 (12,13). Group III consists of FANCD1 (BRCA2), FANCN (PALB2), and FANCJ (BRIP1), which do not play a role in FANCD2 monoubiquitination. BRCA2 regulates formation of RAD51 nucleoprotein filaments during homologous recombination (14, 15), PALB2 is necessary for the correct association of BRCA2 with chromatin (16), and BRIP1 is a BRCA1-associated DNA helicase that contributes to homologous recombination and cross-link repair (17, 18).The FA core complex protein FANCM can specifically bind to model replication forks and Holliday junctions and move the...