DNA Structure-Induced Recruitment and Activation of the Fanconi Anemia Pathway Protein FANCD2

Sobeck, Alexandra; Stone, Stacie; Hoatlin, Maureen E.

doi:10.1128/mcb.02196-06

Cited by 36 publications

(55 citation statements)

References 54 publications

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“…This result is consistent with the findings of Meetei et al (8) showing that in untreated human cells, FANCM exists as a phosphoprotein and is further phosphorylated in response to DNA damage. Moreover, our result supports the idea that the addition of circular plasmid to egg extracts at sufficiently high concentrations mimics the occurrence of DNA damage and triggers a DNA damage response that is uncoupled from ongoing replication (16).…”

Section: Resultssupporting

confidence: 75%

“…Using Xenopus egg extracts, we recently showed that although xFANCD2-Ub is recruited to chromatin in a replication-dependent manner, we can bypass the need for ongoing replication to trigger xFANCD2 monoubiquitination by adding dsDNA substrates, including circular double-stranded DNA (plasmid DNA), to egg extracts (16). We tested whether xFANCM is also modified in response to plasmid DNA.…”

Section: Resultsmentioning

confidence: 99%

“…For DNA damage structures, the preparation of oligonucleotide-derived DNA structures was done as described previously (16). For bead DNA, streptavidin-conjugated magnetic beads (Dynal, Inc.) coupled to biotinylated DNA structures were used for incubation in egg extracts and analysis of DNA structurebound proteins as described before (16).…”

Section: Methodsmentioning

confidence: 99%

“…Tautomycin (3 M) and recombinant Xenopus geminin (5 g/l) were added as indicated. Plasmid DNA was used at 150 ng/l, as described before (16).…”

Section: Methodsmentioning

confidence: 99%

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The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Sobeck¹,

Stone

Landais

et al. 2009

Journal of Biological Chemistry

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In addition to monoubiquitination by the FA core complex, FANCD2 and FANCI are phosphorylated by the two major cell cycle checkpoint kinases, ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related),y in response to DNA damage (2-6). ATM-dependent phosphorylation of FANCD2 occurs following ionizing irradiation and is required for activation of the ionizing irradiation-induced intra-S phase checkpoint (4). ATR-dependent phosphorylation of FANCD2 is triggered by various types of DNA damage, including replication stress, and is required for the interstrand cross-link-induced intra-S phase checkpoint response (2, 3). Moreover, phosphorylation by ATR is required for efficient FANCD2 monoubiquitination in response to DNA damage, suggesting that the FA pathway might participate in ATR-dependent coordination of the S phase of the cell cycle (3, 7).The recent identification of a highly conserved FA core complex member, FANCM (8, 9), indicates a direct role of FA pathway proteins in repair steps at sites of DNA damage. FANCM is a homolog of the archaebacterial Hef protein (helicase-associated endonuclease for fork-structured DNA) and contains two DNA processing domains: a DEAH box helicase domain and an XPF/ERCC4-like endonuclease domain. FANCM has ATP-dependent DNA translocase activity and can dissociate DNA triple helices in vitro (8). Moreover, FANCM binds Holliday junctions and DNA replication fork structures in vitro and promotes ATP-dependent branch point migration, suggesting that FANCM might be involved in DNA processing at stalled replication forks (10,11). In human cells, FANCM localizes to chromatin and is required for chromatin recruitment of other FA core complex proteins (8,12). FANCM is phosphorylated during both the M and S phases and in response to DNA-damaging agents (8,12,13). Interestingly, DNA damage-induced phosphorylation of FANCM is independent of the FA core complex (8), suggesting that FANCM is controlled by other, as yet unknown upstream components of the DNA damage response. Here, we used cell-free Xenopus egg extracts to investigate the role of FANCM during replication and in the DNA damage response. We show that Xenopus FANCM (xFANCM) binds chromatin in a replication-dependent manner and is phosphorylated during unperturbed replication as well as in response to various DNA damage structures. Both chromatin recruitment and phosphorylation of xFANCM are partially controlled by xFANCD2, suggesting feedback signaling from xFANCD2 to the upstream xFA core complex via regulation of xFANCM. In addition, chromatin recruitment during unperturbed replication and activation of xFANCM in response to DNA damage are controlled by the xATR and xATM cell cycle kinases.

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Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Tautomycin (3 M) and recombinant Xenopus geminin (5 g/l) were added as indicated. Plasmid DNA was used at 150 ng/l, as described before (16).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Sobeck¹,

Stone

Landais

et al. 2009

Journal of Biological Chemistry

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show abstract

“…Some researchers found that the FA complex might facilitate the recruitment of other proteins to maintain genomic integrity in the nucleus [37]. The mono-ubiquitinated form of FANCD2, as the central FA pathway protein, is essential for triggering the FA pathway [38]. Moreover, there is also evidence that FANCD2 interacts specifically with TNFR [35].…”

Section: Dan-jun Ma Et Al 657mentioning

confidence: 99%

Temporal and spatial profiling of nuclei-associated proteins upon TNF-α/NF-κB signaling

Wang

et al. 2009

Cell Res

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A novel cell‐free screen identifies a potent inhibitor of the Fanconi anemia pathway

Landais

Sobeck

Stone

et al. 2008

Intl Journal of Cancer

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The Fanconi Anemia (FA) DNA damage response pathway is involved in the processing of DNA interstrand crosslinks (ICLs). As such, inhibition of the FA pathway could chemosensitize FAcompetent tumor cells to commonly used ICL agents like cisplatin. Moreover, suppression of the FA pathway is synthetic lethal with deficiencies in several other DNA repair pathways, suggesting that FA pathway inhibitors could be used in targeted therapies against specific tumors. To identify such inhibitors, we designed a novel in vitro screening assay utilizing Xenopus egg extracts. Using the DNA-stimulated monoubiquitylation of Xenopus FANCD2 (xFANCD2-L) as readout, a chemical library screen identified DDN (2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone) as a novel and potent FA pathway inhibitor. DDN inhibited xFANCD2-L formation in a dose-dependent manner in both extracts and human cells without disruption of the upstream FA core complex. DDN also inhibited the characteristic subnuclear FANCD2 foci formation following DNA damage. Moreover, DDN displayed a greater synergistic effect with cisplatin in a FA-proficient cancer cell line compared to its FA-deficient isogenic counterpart, suggesting that DDN might be a good lead candidate as cisplatin chemosensitizer in both FA-deficient and FA-competent tumors. This system constitutes the first cell-free screening assay for identifying compounds that inhibit the FA pathway and provides a new biochemical platform for mapping the functions of its various components with specific chemical inhibitors. ' 2008 Wiley-Liss, Inc.Key words: chemosensitization; DNA repair; interstrand crosslinks; cisplatin DNA interstrand crosslink (ICL)-inducing agents such as cisplatin, melphalan and mitomycin C (MMC) remain a mainstay in the treatment of cancers such as multiple myeloma, ovarian, testis, breast, bladder and head and neck cancers. 1-5 However, many cancers become refractory to these drugs by multiple mechanisms during a course of chemotherapy, leading to treatment failure. Resensitizing tumor cells to ICL-inducing agents has great potential for treating a wide variety of cancers. One strategy relies on the discovery of small-molecule inhibitors of proteins that control the cellular response to DNA ICLs. Current evidence suggests that the Fanconi Anemia (FA) DNA damage response pathway (which includes breast cancer susceptibility gene products BRCA2, BRIP1/BACH1 and PALB2/FANCN) is involved in the repair of DNA ICLs. As a consequence, acquired ICL-resistance of cancer cells may be associated with re-activation of the FA pathway in tumors initially deficient in this pathway. 3,6,7 Supporting this idea, epigenetic silencing of FANCF has been shown to occur sporadically in AML 7 and in 17, 21 and 30% of breast, ovarian and cervical cancers, respectively. 8 Loss of FANCD2 expression has also been linked with sporadic breast cancer. 5 Interestingly, a recent report by Kennedy et al. identified a set of DNA repair genes [including ataxia telangectasia-mutated (ATM) and NBS1] that are synthetic lethal w...

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DNA Structure-Induced Recruitment and Activation of the Fanconi Anemia Pathway Protein FANCD2

Cited by 36 publications

References 54 publications

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

The Fanconi Anemia Protein FANCM Is Controlled by FANCD2 and the ATR/ATM Pathways

Temporal and spatial profiling of nuclei-associated proteins upon TNF-α/NF-κB signaling

A novel cell‐free screen identifies a potent inhibitor of the Fanconi anemia pathway

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