Niedzwiedz W, Mosedale G, Johnson M, Ong CY, Pace P, Patel KJ.. The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair. Mol Cell 15: 607-620

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
Molecular Cell (Impact Factor: 14.02). 09/2004; 15(4):607-20. DOI: 10.1016/j.molcel.2004.08.009
Source: PubMed


The Fanconi anemia (FA) protein FANCC is essential for chromosome stability in vertebrate cells, a feature underscored by the extreme sensitivity of FANCC-deficient cells to agents that crosslink DNA. However, it is not known how this FA protein facilitates the repair of both endogenously acquired and mutagen-induced DNA damage. Here, we use the model vertebrate cell line DT40 to address this question. We discover that apart from functioning in homologous recombination, FANCC also promotes the mutational repair of endogenously generated abasic sites. Moreover in these vertebrate cells, the efficient repair of crosslinks requires the combined functions of FANCC, translesion synthesis, and homologous recombination. These studies reveal that the FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA.

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    • "Analyses of DNA Fibers, SCEs, and Chromosomal Aberrations Cells were prepared for analyses of DNA fibers, SCEs, and chromosomal aberrations as described (Niedzwiedz et al., 2004; Schwab et al., 2010). "
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    ABSTRACT: The Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 03/2015; 57(6):1133-41. DOI:10.1016/j.molcel.2015.02.012 · 14.02 Impact Factor
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    • "A future challenge is to determine how the various genome maintenance functions of BRCA1 contribute to tumor suppression . Interestingly, the crosslink sensitivity of BRCA1-deficient cells is less severe than for those carrying mutations in other ICL repair factors (Bridge et al., 2005; Niedzwiedz et al., 2004; Ohashi et al., 2005; Qing et al., 2011). This could be explained by the fact that a polymerase-linked mechanism can promote helicase unloading in the absence of BRCA1. "
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    ABSTRACT: The tumor suppressor protein BRCA1 promotes homologous recombination (HR), a high-fidelity mechanism to repair DNA double-strand breaks (DSBs) that arise during normal replication and in response to DNA-damaging agents. Recent genetic experiments indicate that BRCA1 also performs an HR-independent function during the repair of DNA interstrand crosslinks (ICLs). Here we show that BRCA1 is required to unload the CMG helicase complex from chromatin after replication forks collide with an ICL. Eviction of the stalled helicase allows leading strands to be extended toward the ICL, followed by endonucleolytic processing of the crosslink, lesion bypass, and DSB repair. Our results identify BRCA1-dependent helicase unloading as a critical, early event in ICL repair.
    Molecular Cell 09/2014; 56(1). DOI:10.1016/j.molcel.2014.08.012 · 14.02 Impact Factor
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    • "We developed a purification strategy to isolate the FA core complex from chicken DT40 cells, a vertebrate model used extensively in the study of the FA pathway (Niedzwiedz et al., 2004; Yamamoto et al., 2003). Our aim was to generate a cell line where the only copy of the FANCB subunit included an affinity purification tag (Figure 1A). "
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    ABSTRACT: Fanconi anaemia (FA) is a cancer predisposition syndrome characterized by cellular sensitivity to DNA interstrand crosslinkers. The molecular defect in FA is an impaired DNA repair pathway. The critical event in activating this pathway is monoubiquitination of FANCD2. In vivo, a multisubunit FA core complex catalyzes this step, but its mechanism is unclear. Here, we report purification of a native avian FA core complex and biochemical reconstitution of FANCD2 monoubiquitination. This demonstrates that the catalytic FANCL E3 ligase subunit must be embedded within the complex for maximal activity and site specificity. We genetically and biochemically define a minimal subcomplex comprising just three proteins (FANCB, FANCL, and FAAP100) that functions as the monoubiquitination module. Residual FANCD2 monoubiquitination activity is retained in cells defective for other FA core complex subunits. This work describes the in vitro reconstitution and characterization of this multisubunit monoubiquitin E3 ligase, providing key insight into the conserved FA DNA repair pathway.
    Molecular Cell 06/2014; 54(5):858-869. DOI:10.1016/j.molcel.2014.05.001 · 14.02 Impact Factor
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