Differential Phosphorylation of FANCA and FANCG Determines Response of Fanconi Anemia Core Complex to DNA Damage and S Phase.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 726-726
Author(s):  
Jun Mi ◽  
Andrei Tomashevski ◽  
Gary M. Kupfer
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Cellular Responses ◽  
S Phase ◽  
Congenital Defects ◽  
Core Complex ◽  
Downstream Target ◽  
Chk1 Kinase ◽  
Differential Phosphorylation

Abstract Fanconi anemia (FA) is a genetic disease marked by bone marrow failure, congenital defects, and cancer. In spite of the identification of at least 8 genes, the biochemistry of the disease and its normal pathway in the cell remains elusive. The FA core complex is composed of at least 5 proteins, 2 of which, FANCA and FANCG, we have shown to be phosphorylated. In these studies, we show that both FANCA and FANCG are phosphorylated in response to DNA damage. In the case of FANCG, we have mapped the site of this phosphorylation to serine 7, using a phosphoserine 7 FANCG antiserum. Because of the link of FA function and the FA core complex-dependent monoubiquitination that occurs both as a result of DNA damage as well as at S phase, we also examined if phosphorylation occurred at S phase as well. While FANCG serine 7 phosphorylation occurs both at S phase and after DNA damage (similar to FANCD2 monoubiquitination), FANCA phosphorylation occurs only after DNA damage. Recent data have implicated the kinase ATR as important in the pathway. In order to assess whether a downstream target of ATR is differentially phosphorylated in FA cells, we tested the phosphorylation status of chk1 in FA-A mutant and corrected cells. Chk1 kinase is phosphorylated at serine 318 in response to DNA damage only in corrected cells but not mutant FA cells, while signaling through chk2 kinase is unaffected. These data suggest the importance of phosphorylation in the FA pathway in the regulation of both cellular responses to DNA damage as well as engagement of the cell cycle.

Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 723-723
Author(s):  
Alexandra Sobeck ◽  
Stacie Stone ◽  
Bendert deGraaf ◽  
Vincenzo Costanzo ◽  
Johan deWinter ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
S Phase ◽  
Dependent Manner ◽  
Core Complex ◽  
Damage Response ◽  
Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

scholarly journals Evidence for subcomplexes in the Fanconi anemia pathway

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 2072-2080 ◽  
Author(s):  
Annette L. Medhurst ◽  
El Houari Laghmani ◽  
Jurgen Steltenpool ◽  
Miriam Ferrer ◽  
Chantal Fontaine ◽  
...  
Keyword(s):  
Fanconi Anemia ◽  
Protein Interactions ◽  
Congenital Abnormalities ◽  
Bone Marrow Failure ◽  
Direct Interaction ◽  
Molecular Architecture ◽  
Cross Link ◽  
Core Complex ◽  
Downstream Target ◽  
Nuclear Complex

AbstractFanconi anemia (FA) is a genomic instability disorder, clinically characterized by congenital abnormalities, progressive bone marrow failure, and predisposition to malignancy. Cells derived from patients with FA display a marked sensitivity to DNA cross-linking agents, such as mitomycin C (MMC). This observation has led to the hypothesis that the proteins defective in FA are involved in the sensing or repair of interstrand cross-link lesions of the DNA. A nuclear complex consisting of a majority of the FA proteins plays a crucial role in this process and is required for the monoubiquitination of a downstream target, FANCD2. Two new FA genes, FANCB and FANCL, have recently been identified, and their discovery has allowed a more detailed study into the molecular architecture of the FA pathway. We demonstrate a direct interaction between FANCB and FANCL and that a complex of these proteins binds FANCA. The interaction between FANCA and FANCL is dependent on FANCB, FANCG, and FANCM, but independent of FANCC, FANCE, and FANCF. These findings provide a framework for the protein interactions that occur “upstream” in the FA pathway and suggest that besides the FA core complex different subcomplexes exist that may have specific functions other than the monoubiquitination of FANCD2.

ATR Dependent Phosphorylation of FANCM Is Required for the Fanconi Anemia Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 837-837
Author(s):  
Thiyam R. Singh ◽  
Abdullah M. Ali ◽  
Chang-hu Du ◽  
Ruhikanta A. Meetei
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Phosphorylation Site ◽  
Cell Cycle Checkpoint ◽  
Phosphorylation Sites ◽  
Core Complex ◽  
Knock Out ◽  
Hct116 Cells

Abstract Fanconi anemia (FA) is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. We have shown that FANCM which is one of the FA core complex proteins is hyperphosphorylated in response to DNA damage suggesting that it may serve as a signal transducer through which the activity of the FA-core complex is regulated. The cell cycle checkpoint kinase, ATR has been shown to act upstream of the FA pathway, however, its substrate within the FA-core complex has not been identified yet. FANCM contains multiple predicted ATR phosphorylation sites suggesting that FANCM could be a direct ATR target. In this study, we examined the roles of ATR in regulating FANCM phosphorylation in response to DNA damage: by kinetics study we found that phosphorylation of FANCM is concurrent with FANCD2 monoubiquitination; siRNA mediated suppression of ATR activity abrogates both phosphorylation of FANCM and monoubiquitination of FANCD2; and ATR knock out HCT116 cells display defective phosphorylation of FANCM as well as defective monoubiquitination of FANCD2 indicating that DNA damage induced phosphorylation of FANCM is ATR dependant. Furthermore, we used mass spectrometry to identify the in vivo phosphorylation sites of FANCM and found a novel DNA damage-inducible phosphorylation site (S-1045; one of the potential ATR phosphorylation sites) within FANCM protein. Using ATR knock out HCT116 cells and the anti-p-S1045 antibody, we show that phosphorylation of FANCM at S-1045 is ATR dependant. The biological relevance of phosphorylation of FANCM at S1045 in FA pathway will be investigated by functional complementation analysis with non phosphorylatable FANCM mutants in FANCM deficient cells.

scholarly journals UBE2T, the Fanconi Anemia Core Complex, and FANCD2 Are Recruited Independently to Chromatin: a Basis for the Regulation of FANCD2 Monoubiquitination

2007 ◽  
Vol 27 (24) ◽  
pp. 8421-8430 ◽  
Author(s):  
Arno Alpi ◽  
Frederic Langevin ◽  
Georgina Mosedale ◽  
Yuichi J. Machida ◽  
Anindya Dutta ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Fanconi Anemia ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Multiprotein Complex ◽  
Core Complex ◽  
Nuclear Core ◽  
Ubiquitin Conjugating Enzyme ◽  
Fancd2 Monoubiquitination

ABSTRACT The Fanconi anemia (FA) nuclear core complex and the E2 ubiquitin-conjugating enzyme UBE2T are required for the S phase and DNA damage-restricted monoubiquitination of FANCD2. This constitutes a key step in the FA tumor suppressor pathway, and much attention has been focused on the regulation at this point. Here, we address the importance of the assembly of the FA core complex and the subcellular localization of UBE2T in the regulation of FANCD2 monoubiquitination. We establish three points. First, the stable assembly of the FA core complex can be dissociated of its ability to function as an E3 ubiquitin ligase. Second, the actual E3 ligase activity is not determined by the assembly of the FA core complex but rather by its DNA damage-induced localization to chromatin. Finally, UBE2T and FANCD2 access this subcellular fraction independently of the FA core complex. FANCD2 monoubiquitination is therefore not regulated by multiprotein complex assembly but by the formation of an active E2/E3 holoenzyme on chromatin.

The Fanconi anemia core complex associates with chromatin during S phase

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 759-766 ◽  
Author(s):  
Jun Mi ◽  
Gary M. Kupfer
Keyword(s):  
Fanconi Anemia ◽  
Autosomal Recessive ◽  
Autosomal Recessive Disease ◽  
Bone Marrow Failure ◽  
Nuclear Periphery ◽  
S Phase ◽  
Core Complex ◽  
Fiber Analysis ◽  
Chromatin Fibers ◽  
Complex Movement

AbstractFanconi anemia (FA) is an autosomal recessive disease marked by bone marrow failure, birth defects, and cancer. The FA proteins FANCA, FANCC, FANCE, FANCF, FANCG, and FANCL participate in a core complex. We previously have shown that several members of this complex bind to chromatin until mitosis and that this binding increases after DNA damage. The purpose of the present study was to determine the dynamics of complex movement between cytoplasm and nuclear compartments. Fluorescent-tagged versions of FANCA, FANCC, and FANCG colocalize in cytoplasm and nucleus, chiefly in chromatin. At the G1-S border, the FA core complex exists as foci on chromatin, progressively diffusing and migrating to the nuclear periphery and becoming completely excluded from condensed chromosomes by mitosis. Chromatin fiber analysis shows FA proteins diffusely staining along chromatin fibers during G1-S and S phase. Treatment with the DNA cross-linker mitomycin C results in a diffusion of foci and increased binding of complex proteins to chromatin, as well as diffuse and increased complex binding to chromatin fibers. These data are consistent with the idea that the FA proteins function at the level of chromatin during S phase to regulate and maintain genomic stability.

scholarly journals Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair

2014 ◽  
Vol 42 (15) ◽  
pp. 9807-9820 ◽  
Author(s):  
Meghan Larin ◽  
David Gallo ◽  
Laura Tamblyn ◽  
Jay Yang ◽  
Hudson Liao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Congenital Abnormalities ◽  
Bone Marrow Failure ◽  
In Utero ◽  
Critical Threshold ◽  
Growth Defects ◽  
Repair Defect ◽  
In Utero Development ◽  
Crosslink Repair

AbstractIndividuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair. The relationship of this repair defect to disease traits remains unclear, given that crosslink sensitivity is recapitulated in FA mouse models without most of the other disease-related features. Mice deficient in Mus81 are also defective in crosslink repair, yet MUS81 mutations have not been linked to FA. Using mice deficient in both Mus81 and the FA pathway protein FancC, we show both proteins cooperate in parallel pathways, as concomitant loss of FancC and Mus81 triggered cell-type-specific proliferation arrest, apoptosis and DNA damage accumulation in utero. Mice deficient in both FancC and Mus81 that survived to birth exhibited growth defects and an increased incidence of congenital abnormalities. This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity. Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.

scholarly journals DNA Repair: Exploiting the Fanconi Anemia Pathway As a Potential Therapeutic Target

2011 ◽  
pp. 453-465 ◽  
Author(s):  
T. HUCL ◽  
E. GALLMEIER
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Fanconi Anemia ◽  
Cancer Susceptibility ◽  
Synthetic Lethality ◽  
Bone Marrow Failure ◽  
Long Term Effects ◽  
Alternative Pathways ◽  
Repair Mechanisms ◽  
Relationship Of

DNA repair is an active cellular process to respond to constant DNA damage caused by metabolic processes and environmental factors. Since the outcome of DNA damage is generally adverse and long term effects may contribute to oncogenesis, cells have developed a variety of DNA repair mechanisms, which operate depending on the type of DNA damage inflicted. At least 15 Fanconi anemia (FA) proteins interact in a common pathway involved in homologous recombination. Inherited homozygous mutations in any of these FA genes cause a rare disease, Fanconi anemia, characterized by congenital abnormalities, progressive bone-marrow failure and cancer susceptibility. Heterozygous germline FA mutations predispose to various types of cancer. In addition, somatic FA mutations have been identified in diverse cancer types. Evidence exists that cells deficient in the FA pathway become dependent on alternative pathways for survival. Additional inhibition of such alternative pathways is thus expected to result in cell death, creating a relationship of synthetic lethality. Identifying these relationships can reveal yet unknown mechanisms of DNA repair and new targets for therapy.

scholarly journals Holding All the Cards—How Fanconi Anemia Proteins Deal with Replication Stress and Preserve Genomic Stability

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 170 ◽  
Author(s):  
Arindam Datta ◽  
Robert M. Brosh Jr.
Keyword(s):  
Dna Damage ◽  
Fanconi Anemia ◽  
Excision Repair ◽  
Double Helix ◽  
Bone Marrow Failure ◽  
Molecular Genetic ◽  
Replication Stress ◽  
Gene Products ◽  
Hematopoietic Stem ◽  
Dna Double Helix

Fanconi anemia (FA) is a hereditary chromosomal instability disorder often displaying congenital abnormalities and characterized by a predisposition to progressive bone marrow failure (BMF) and cancer. Over the last 25 years since the discovery of the first linkage of genetic mutations to FA, its molecular genetic landscape has expanded tremendously as it became apparent that FA is a disease characterized by a defect in a specific DNA repair pathway responsible for the correction of covalent cross-links between the two complementary strands of the DNA double helix. This pathway has become increasingly complex, with the discovery of now over 20 FA-linked genes implicated in interstrand cross-link (ICL) repair. Moreover, gene products known to be involved in double-strand break (DSB) repair, mismatch repair (MMR), and nucleotide excision repair (NER) play roles in the ICL response and repair of associated DNA damage. While ICL repair is predominantly coupled with DNA replication, it also can occur in non-replicating cells. DNA damage accumulation and hematopoietic stem cell failure are thought to contribute to the increased inflammation and oxidative stress prevalent in FA. Adding to its confounding nature, certain FA gene products are also engaged in the response to replication stress, caused endogenously or by agents other than ICL-inducing drugs. In this review, we discuss the mechanistic aspects of the FA pathway and the molecular defects leading to elevated replication stress believed to underlie the cellular phenotypes and clinical features of FA.

Xeroderma Pigmentosum as a Model for Treatment of Bone Marrow Failure in Fanconi Anemia and Aplastic Anemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4235-4235
Author(s):  
W. Clark Lambert ◽  
Santiago A. Centurion
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Fanconi Anemia ◽  
Xeroderma Pigmentosum ◽  
Bone Marrow Failure ◽  
S Phase ◽  
White Female ◽  
Cross Linking

Abstract We have previously shown that the primary cell cycle defect in the inherited, cancer-prone, bone marrow failure associated disease, Fanconi anemia (FA), is not in the G2 phase of the cell cycle, as had been thought for many years, but rather in the S phase. FA cells challenged with the DNA cross-linking agent, psoralen coupled with long wavelength, ultraviolet (UVA) radiation (PUVA), fail to slow their progression through the S phase of the subsequent cell cycle, as do normal cells. FA cells are extremely sensitive to the cytotoxic and clastogenic effects of DNA cross-linkers, such as PUVA, so much so that the diagnosis of FA is based on an assay, the “DEB test”, in which cells are examined for clastogenic and cytotoxic effects of diepoxybutane (DEB), a DNA cross-linking agent. More recently, we have shown that artificially slowing the cell cycle of FA cells exposed to PUVA by subsequent treatment with agents which slow their progression through S phase leads to markedly increased viability and reduced chromosome breakage in vitro. We now show that similar results can be obtained in vivo in patients with another DNA repair deficiency disease, xeroderma pigmentosum (XP), a recessively inherited disorder associated with defective repair of sunlight induced adducts in the DNA of sun-exposed tissues followed by development of numerous mutations causing large numbers of cancers in these same tissues. We treated two patients with XP, a light complected black male and a white female, both 14 years of age, in sun-exposed areas with 5-fluorouracil, an inhibitor of DNA synthesis, daily for three months. In contrast to normal patients, who only show clinical results if an inflammatory response is invoked, marked improvement in the clinical appearance of the skin was seen with no inflammation observed. This effect was confirmed histologically by examining epidermis adjacent to excised lesions in sun-exposed areas and further verified by computerized image analysis. Treatment with agents that slow progression through S phase, such as hydroxyurea, may similarly improve clinical outcomes in patients with FA or others who are developing bone marrow failure.

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