In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5-THOC7, which have no orthologs in budding yeast. Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO. Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function. Notably, 8% of upregulated genes encode proteins involved in DNA repair. Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D. melanogaster.

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.

The function of many DNA metabolism proteins depends on their ability to coordinate an iron-sulfur (Fe-S) cluster. Biogenesis of Fe-S proteins is a multistep process that takes place in mitochondria and the cytoplasm, but how it is linked to nuclear Fe-S proteins is not known. Here, we demonstrate that MMS19 forms a complex with the cytoplasmic Fe-S assembly (CIA) proteins CIAO1, IOP1, and MIP18. Cytoplasmic MMS19 also binds to multiple nuclear Fe-S proteins involved in DNA metabolism. In the absence of MMS19, a failure to transfer Fe-S clusters to target proteins is associated with Fe-S protein instability and preimplantation death of mice in which Mms19 has been knocked out. We propose that MMS19 functions as a platform to facilitate Fe-S cluster transfer to proteins critical for DNA replication and repair.

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.

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...

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.

SummaryThe cytoplasmic iron-sulfur assembly (CIA) targeting complex is required for the transfer of an iron-sulfur (Fe-S) cluster to cytoplasmic and nuclear proteins, but how it engages with client proteins is unknown. Here, we show that the complex members MIP18 and CIAO1 associate with the C terminus of MMS19. By doing so, they form a docking site for Fe-S proteins that is disrupted in the absence of either MMS19 or MIP18. The Fe-S helicase XPD seems to be the only exception, since it can interact with MMS19 independently of MIP18 and CIAO1. We further show that the direct interaction between MMS19 and MIP18 is required to protect MIP18 from proteasomal degradation. Taken together, these data suggest a remarkably regulated interaction between the CIA targeting complex and client proteins and raise the possibility that Fe-S cluster transfer is controlled, at least in part, by the stability of the CIA targeting complex itself.

SummaryDuring DNA replication stress, stalled replication forks need to be stabilized to prevent fork collapse and genome instability. The AAA + ATPase WRNIP1 (Werner Helicase Interacting Protein 1) has been implicated in the protection of stalled replication forks from nucleolytic degradation, but the underlying molecular mechanism has remained unclear. Here we show that WRNIP1 exerts its protective function downstream of fork reversal. Unexpectedly though, WRNIP1 is not part of the well-studied BRCA2-dependent branch of fork protection but seems to protect the junction point of reversed replication forks from SLX4-mediated endonucleolytic degradation, possibly by directly binding to reversed replication forks. This function is specific to the shorter, less abundant, and less conserved variant of WRNIP1. Overall, our data suggest that in the absence of BRCA2 and WRNIP1 different DNA substrates are generated at reversed forks but that nascent strand degradation in both cases depends on the activity of exonucleases and structure-specific endonucleases.