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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Available from: Takamune T. Saito
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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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    • "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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    • "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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