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: 14.46). 07/2010; 39(1):36-47. DOI: 10.1016/j.molcel.2010.06.023
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Takamune T. Saito, Aug 30, 2015
  • Source
    • "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 We manually curated a list of 726 candidate genes (851 open reading frames [ORFs]) using published and unpublished datasets relevant to the DDR, including a set of genes that encode proteins experimentally identified to be chromatin-enriched after exposure to DNA damage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). "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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 · 2.85 Impact Factor
  • Source
    • "The structure of PaFAN1 bound to 59 flap DNA reveals that the four domains of FAN1 lacking a UBZ domain are sufficient and essential for the recognition of flap DNA. This is consistent with previous studies demonstrating that removal of other domains notably diminishes its localization to the lesion (Liu et al. 2010; Smogorzewska et al. 2010). During ICL repair, FAN1 is expected to be anchored to the lesion through the monoubiquitinated ID complex (R€ aschle et al. 2008; Knipscheer et al. 2009). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5′ flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5′ flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.
    Genes & development 09/2014; 28(28):2276-2290. DOI:10.1101/gad.248492.114 · 12.64 Impact Factor
Show more