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FAN1 Acts with FANCI-FANCD2 to Promote DNA Interstrand Cross-Link Repair

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Science  06 Aug 2010:
Vol. 329, Issue 5992, pp. 693-696
DOI: 10.1126/science.1192656

Abstract

Fanconi anemia (FA) is caused by mutations in 13 Fanc genes and renders cells hypersensitive to DNA interstrand cross-linking (ICL) agents. A central event in the FA pathway is mono-ubiquitylation of the FANCI-FANCD2 (ID) protein complex. Here, we characterize a previously unrecognized nuclease, Fanconi anemia–associated nuclease 1 (FAN1), that promotes ICL repair in a manner strictly dependent on its ability to accumulate at or near sites of DNA damage and that relies on mono-ubiquitylation of the ID complex. Thus, the mono-ubiquitylated ID complex recruits the downstream repair protein FAN1 and facilitates the repair of DNA interstrand cross-links.

Fanconi anemia (FA) is characterized by congenital malformations, bone marrow failure, cancer, and hypersensitivity to DNA interstrand cross-linking (ICL) agents (13). Resistance to DNA ICL agents probably requires all FA proteins (4, 5). Eight FA proteins (A, B, C, E, F, G, L, and M) are assembled into the nuclear FA core complex that mono-ubiquitylates its two substrates, FANCI and FANCD2 (410), which, in turn, form DNA damage–induced nuclear foci together with other key DNA damage–response proteins (1, 4). Failure to mono-ubiquitylate FANCI and FANCD2 results in highly decreased efficiency of DNA cross-link repair (4). The mono-ubiquitylated FANCI-FANCD2 (ID) complex might promote the recognition and subsequent removal of DNA lesions through nucleolytic cleavage of DNA strands by recruitment of ubiquitin-binding proteins that are important for this repair process (1116). DNA damage–response and –repair proteins can be recruited to sites of DNA damage via their ubiquitin-binding domains (1720). We identified a protein, KIAA1018, that contains a single ubiquitin-binding zinc finger (ZNF) domain at its N terminus and a virus-type replication-repair (VRR)–nuclease domain (or DUF994 domain) (21, 22) at its C terminus (fig. S1, A and B). KIAA1018 relocalized to damage-induced foci after mitomycin C (MMC) treatment (fig. S1, C to F), suggesting that this protein is involved in DNA damage response. Because of the functional analyses performed below, we designated this protein as Fanconi anemia–associated nuclease 1 (FAN1).

Proteins associated with FAN1 were identified by mass spectrometry in a human embryonic kidney 293T–derivative cell line stably expressing a triple-tagged FAN1 (23). We repeatedly found the ID complex as major FAN1-associated proteins (fig. S2A). Mass spectrometry analyses of triple-tagged FANCD2-associated protein complexes revealed peptides that corresponded to FAN1 (fig. S2B), indicating that these proteins probably form a complex in vivo. Immunoprecipitation (IP) confirmed the interaction of FAN1 with FANCD2 and weakly with FANCI (Fig. 1A). Although FAN1 could interact with the unmodified ID complex, its association with the ID complex was greatly enhanced after MMC treatment (Fig. 1B), which coincides with FANCI-FANCD2 mono-ubiquitylation (68). Their association was further confirmed by in vivo colocalization experiments (fig. S2, C and D).

Fig. 1

FAN1 acts in ICL repair downstream of FANCD2/I. (A) Ectopically expressed FAN1 interacts with FANCD2/FANCI. 293T cells were cotransfected with plasmids encoding Myc-tagged FAN1 and S, FLAG, and streptavidin-binding peptide tag (SFB)–tagged FANCD2 or FANCI. IP reactions were done using the antibodies as indicated. (B) Interaction between FAN1 and FANCD2/I before and after MMC treatment was monitored by IP with an antibody to FAN1 (anti-FAN1) and detected on SDS–polyacrylamide gel electrophoresis (PAGE) gels with the indicated antibodies (top three panels). IgG, immunoglobulin G. (C) FAN1 is not required for FANCD2/I mono-ubiquitylation. Soluble and chromatin fractions prepared from mock-treated or MMC-treated HeLa cells and immunoblotting experiments were performed using the indicated antibodies. W, whole-cell extracts; SiCon, control siRNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H3, histone H3. (D) FANCD2/I is required for FAN1 foci formation. HeLa cells were transfected with either FANCD2/I siRNAs or control siRNA and then treated with 1 μM MMC for 24 hours before immunostaining experiments were performed. DAPI, 4′,6′-diamidino-2-phenylindole. (E) FANCD2/I is required for FAN1 chromatin recruitment. Chromatin fractions were isolated, and immunoblotting experiments were performed using the indicated antibodies. (F and G) FAN1-depleted cells display increased MMC sensitivity. These experiments were performed in triplicate, and the results were the average of three independent experiments. The SD is shown for different doses of MMC or irradiation.

Upon reduction of endogenous FAN1 expression, we still detected MMC-induced mono-ubiquitylation and foci formation of FANCI-FANCD2 (Fig. 1C and fig. S3, A and B). Upon depletion of FANCD2 or FANCI, we failed to observe FAN1 foci after MMC treatment and saw a substantially reduced chromatin accumulation of FAN1 after MMC treatment (Fig. 1, D and E, and fig. S3C). Knockdown of FAN1 caused a significant increase in MMC but not camptothecin sensitivity (Fig. 1, F and G, and fig. S3D), increased levels of MMC-induced chromosome instability (fig. S4, A and B), and profound G2/M-phase arrest (fig. S4C), all typical of FA cells (15). Double knockdown of FAN1 with FANCD2 or FANCA did not lead to any further increase in these phenotypes (Fig. 1F and fig. S4). Altogether, FAN1 promotes ICL repair downstream of the ID complex and does so through a common pathway.

The ZNF domain deletion mutant (ΔZNF) of FAN1 lost its foci-formation ability, whereas the nuclease domain mutant (ΔNUC) still localized to nuclear foci after MMC treatment (fig. S5, A to C). An N-terminal fragment, which contains the intact ZNF domain, but not a ZNF domain–disrupting point mutant (ZNF-C44F), is sufficient for foci formation after MMC treatment (fig. S5, A to C). Wild-type (WT) or the ΔNUC mutant of FAN1, but not the FAN1 mutant that lacks its ZNF domain (ΔZNF), specifically interacted with a ubiquitin-glutathione S-transferase fusion protein (Ubi-GST) in vitro (Fig. 2A). In addition, Ubi-GST pulled down the N-terminal fragment of FAN1 containing the ZNF domain, but not the ZNF-C44F mutant (Fig. 2A). WT FAN1, but not FAN1 mutant that lacks its ZNF domain (ΔZNF), interacts with the ID complex, and this interaction is greatly enhanced after MMC treatment (Fig. 2B). WT FANCD2/FANCI interacted strongly with FAN1, whereas the ubiquitylation-deficient point mutants showed only moderate or residual binding (Fig. 2C). Moreover, the N-terminal fragment of FAN1 containing the intact ZNF domain, but not its corresponding ZNF-C44F mutant or the ZNF domain of RAD18 (RAD18-ZNF), interacted with FANCD2 (Fig. 2D). WT FANCD2 could restore FAN1 foci formation in FANCD2-deficient PD20 cells, but the mono-ubiquitylation mutant Lys561 → Arg561 (K561R) (24) of FANCD2 did not (Fig. 2E and fig. S5D). Foci formation of the FAN1 ZNF domain alone also depends on FANCD2 mono-ubiquitylation (Fig. 2F and fig. S5E). The mono-ubiquitylation mutant (K523R) of FANCI partially complemented FAN1 foci formation in FANCI-depleted cells (Fig. 2G and fig. S5F), as mono-ubiquitylation of FANCI is not critical for the function of the FA pathway (8, 25). Thus, mono-ubiquitylated FANCD2 (and FANCI) acts to facilitate FAN1 accumulation at sites of DNA damage.

Fig. 2

Focus-localization of FAN1 depends on its ZNF domain and mono-ubiquitylation of the ID complex. (A) The ZNF domain of FAN1 is essential and sufficient for binding to ubiquitin in vitro. (B) The ZNF domain of FAN1 is required for binding to FANCD2/I. 293T cells stably expressing SFB-tagged FANCD2 or FANCI were transfected with plasmids encoding Myc-tagged WT or ZNF domain–deletion mutant of FAN1. Cells were mock treated or treated with MMC before IP reactions were performed. (C) Mono-ubiquitylation of FANCD2/FANCI is required for binding to FAN1. (D) The ability to bind the mono-ubiquitylated form of FANCD2 is specific to the ZNF domain of FAN1. IP reactions were performed as described in (B). (E and F) Dependence of DNA damage–induced FAN1 foci formation on mono-ubiquitylation of FANCD2. PD20 cells, expressing SFB-tagged WT FANCD2 or the K561R mutant (FANCD2-KR), were treated with MMC (E) or transfected with plasmids encoding hemagglutinin (HA)–tagged FAN1 ZNF domain before MMC treatment (F). Immunostaining experiments were performed as indicated. (G) Partial dependence of damage-induced FAN1 foci formation on mono-ubiquitylation of FANCI. HeLa cells depleted of endogenous FANCI were infected with viruses encoding siRNA-resistant HA-Flag–tagged WT or K523R mutant of FANCI (FANCI-KR). Cells were treated with MMC and immunostained as indicated.

ICL repair involves nucleolytic cleavage at or near the site of ICL to produce a suitable substrate that can subsequently be repaired by homologous recombination (HR) (14). Purified FAN1 (fig. S6, A and B) was incubated with 5′-flap DNA substrate and displayed nuclease domain-dependent endonuclease activity (Fig. 3A). FAN1 could also cleave branched DNA structures (such as splayed-arm, 3′-flap, 5′-flap, or replication-fork structures), but not duplex DNA (Fig. 3B), indicating that FAN1 is a structure-specific endonuclease. To confirm that the nuclease activity we observed is intrinsic to FAN1, we generated FAN1 mutations at two highly conserved residues within its nuclease domain (D960A and K977A). Both of these mutants abolished the endonuclease activity of FAN1 on 5′-flap DNA substrate or other branched DNA substrates (Fig. 3A and fig. S6C).

Fig. 3

FAN1 is a nuclease that promotes ICL repair. (A) FAN1 displays endonuclease activity on 5′-flap DNA substrate. 2.5 nM 5′ 32P-end–labeled 5′-flap DNA (lane 1) was incubated with 25 nM (lanes 2, 5, and 8), 50 nM (lanes 3, 6, and 9), and 100 nM (lanes 4, 7, and 10) of FAN1, ΔZNF, and ΔNUC, respectively, in the presence of Mg2+ at 37°C for 30 min (left). Alternatively, the same 5′-flap DNA substrate (lane 1) was incubated with 50 nM (lanes 2, 4, and 6) and 100 nM (lanes 3, 5, and 7) of D960A, K977A, and WT FAN1, respectively (right). Reaction products were analyzed on denaturing PAGE. Asterisks indicate 5′ 32P-end label. (B) FAN1 is a structure-specific endonuclease. Indicated 2.5 nM 5′ 32P-end–labeled DNA substrates (lane 1) were incubated with 1, 5, 10, 25, and 50 nM FAN1 (lanes 2 to 6) in the presence of Mg2+ for 30 min at 37°C. Reaction products were analyzed on denaturing PAGE. (C and D) The ZNF domain and nuclease activity of FAN1 are required for restoring cellular resistance to MMC. HeLa-derivative cell lines stably expressing siRNA-resistant HA-Flag–tagged WT (FAN1SiR-WT), ΔZNF mutant (FAN1SiR-ΔZNF), D960A mutant (FAN1SiR-D960A), and Κ977Α mutant (FAN1SiR-K977A) of FAN1 were generated. FAN1 expression was confirmed by immunoblotting with the use of Flag antibody, and extracts were prepared from cells transfected with FAN1 siRNA#1 (D). These experiments were performed in triplicate, and the results were the average of three independent experiments (C). The SD is shown for different doses of MMC.

To explore the physiological relevance of this highly conserved nuclease domain and the ZNF domain of FAN1 in ICL repair, we knocked down FAN1 in HeLa cells using siFAN1#1 [FAN1-specific small interfering RNA (siRNA) 1] and reintroduced siRNA-resistant full-length FAN1, ΔZNF, or the nuclease-inactivating mutants (D960A and K977A) of FAN1. Clonogenic assays indicated that reconstitution with WT FAN1, but not its ZNF deletion (D960A or K977A mutant), restored cell survival after MMC treatment (Fig. 3, C and D), suggesting that both the nuclease activity and the ZNF domain of FAN1 are important for FAN1 function in promoting cell survival after MMC treatment.

FAN1 is a nuclease that associates with mono-ubiquitylated FANCI-FANCD2, mutations that may be responsible for FA in a subset of human patients. FAN1 is a structure-specific endonuclease that may act together with other repair proteins to mediate endonucleolytic digestion of cross-linked DNA structures and, thus, generate ends that can serve as substrates for HR repair.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1192656/DC1

Materials and Methods

SOM Text

Figs. S1 to S8

References

References and Notes

  1. Materials and methods are available as supportnig material on Science Online.
  2. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
  3. This work was supported in part by grants from Startup Fund (Life Sciences Institute, Zhejiang University to J.H.), the Fundamental Research Funds for the Central Universities, China (to J.H.), and the NIH (to J.C.). We thank L. Li, X. Shen, and Y. Zhang for reagents. J.H. would like to thank J.C. for his continuous support and mentoring. J.C. is a recipient of an Era of Hope Scholar award from the U.S. Department of Defense and is a member of M.D. Anderson Cancer Center.
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