A Genetic Screen Identifies FAN1, a Fanconi Anemia-Associated Nuclease Necessary for DNA Interstrand Crosslink Repair

Howard Hughes Medical Institute, Department of Genetics, Harvard Medical School, and Brigham and Women's Hospital, Boston, MA 02115, USA.
Molecular cell (Impact Factor: 14.02). 07/2010; 39(1):36-47. DOI: 10.1016/j.molcel.2010.06.023
Source: PubMed


The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1's ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5'-3' exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.

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    • "The MUS81-EME1 nuclease complex also associates with SLX4 and functions in ICL repair, but is dispensable for initial ICL processing in Xenopus egg extracts (Klein Douwel et al., 2014; Räschle et al., 2015), suggesting that the complex may process different intermediates in ICL repair such as Holliday junction (HJ) resolution during double-strand break (DSB) repair (Chen et al., 2001). FAN1, another structure-specific nuclease implicated in ICL repair, contains a UBZ4 motif required for interaction with FANCD2-Ub (Smogorzewska et al., 2010). However, FAN1 deficiency leads not to FA, but rather to a rare kidney disease called karyomegalic interstitial nephritis (KIN), suggesting that FAN1 may play a redundant role with other nucleases in the FA pathway or deal with lesions independently of the FA pathway (Zhou et al., 2012). "
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    ABSTRACT: Genome instability, primarily caused by faulty DNA repair mechanisms, drives tumorigenesis. Therapeutic interventions that exploit deregulated DNA repair in cancer have made considerable progress by targeting tumor-specific alterations of DNA repair factors, which either induces synthetic lethality or augments the efficacy of conventional chemotherapy and radiotherapy. The study of Fanconi anemia (FA), a rare inherited blood disorder and cancer predisposition syndrome, has been instrumental in understanding the extent to which DNA repair defects contribute to tumorigenesis. The FA pathway functions to resolve blocked replication forks in response to DNA interstrand cross-links (ICLs), and accumulating knowledge of its activation by the ubiquitin-mediated signaling pathway has provided promising therapeutic opportunities for cancer treatment. Here, we discuss recent advances in our understanding of FA pathway regulation and its potential application for designing tailored therapeutics that take advantage of deregulated DNA ICL repair in cancer.
    Moleculer Cells 07/2015; 38(8). DOI:10.14348/molcells.2015.0175 · 2.09 Impact Factor
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    • "RESULTS A Focused Screen to Identify Proteins that Localize to Damaged Chromatin To better understand the complement of proteins that respond to DNA lesions and control localized DNA repair, we conducted a focused screen for proteins that are recruited to DNA breaks induced by UV laser microirradiation (Figure 1A). We manually curated a list of 726 candidate genes (851 open reading frames [ORFs]) using published and unpublished datasets relevant to the DDR, including a set of genes that encode proteins experimentally identified to be chromatin-enriched after exposure to DNA damage (Tables S1, S2, and S3) (Adamson et al., 2012; Chou et al., 2010; Cotta-Ramusino et al., 2011; Hurov et al., 2010; Matsuoka et al., 2007; O'Connell et al., 2010; Paulsen et al., 2009; S1abicki et al., 2010; Smogorzewska et al., 2010). Candidate ORFs were selected from sequence-verified human ORFeome collections and were individually transduced into U2OS cells for expression as N-or C-terminal hemagglutinin (HA) fusion proteins using lentiviruses (Figure 1A). "
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    ABSTRACT: Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 05/2015; 11(9). DOI:10.1016/j.celrep.2015.04.053 · 8.36 Impact Factor
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    • "In 2010, four groups identified a novel FA-associated nuclease, FAN1, that directly binds to monoubiquitinated FANCD 2 , participating in ICL repair (Kratz et al., 2010; Liu et al., 2010; MacKay et al., 2010; Smogorzewska et al., 2010). FAN1 is a structure-specific nuclease and possesses preferential endonuclease activity toward 5′ flap structures and a weaker 5′-3′ exonuclease activity in vitro and has been hypothesized to cleave DNA in multiple steps (MacKay et al., 2010; O'Donnell and Durocher, 2010). "
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    ABSTRACT: Dear Editor,DNA interstrand crosslinks (ICLs) are toxic to cells because they covalently link the two strands in the duplex. ICLs can be induced by crosslinking agents and by cell endogenous metabolites, and physically prevent transcription and replication in both directions (MacKay et al., 2010). This type of DNA damage must be properly repaired to maintain genomic and cellular integrity, and this repair is primarily accomplished by the Fanconi anemia pathway (FA pathway) (Moldovan and D’Andrea, 2009). FA is an inherited recessive developmental and cancer predisposition syndrome that can be caused by defects in any of 16 FANC proteins (Chaudhury et al., 2014). The removal of ICLs depends on numerous components involved in multiple DNA repair pathways such as nucleotide excision repair (NER), mismatch repair (MMR), translesion synthesis (TLS) and homologous recombination (HR), and several models have been previously proposed (Fu et al., 2011; Huang et al., 2013; Kratz et al., 2010). Am ...
    Protein & Cell 12/2014; 6(3). DOI:10.1007/s13238-014-0128-y · 3.25 Impact Factor
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