_:b583719529 "2"^^ . _:b583719506 . _:b583719544 . _:b583719528 "2"^^ . _:b583719517 . _:b583719486 . . _:b583719531 "2"^^ . . . _:b583719516 . _:b42336736 . _:b583719530 "2"^^ . _:b583719519 . _:b583719477 . _:b583719541 "2"^^ . . _:b583719518 . _:b583719540 "2"^^ . _:b583719513 . _:b42336758 "Since Ube2T auto-monoubiquitinates (22), and FANCL has been shown to stimulate this (>>22<<), we performed a thioester discharge assay to test whether the ELF-Ubiquitin interaction is important for this." . _:b583719543 "2"^^ . _:b583719512 . . _:b583719542 "2"^^ . _:b583719515 . . _:b583719537 "2"^^ . _:b583719514 . . _:b583719536 "2"^^ . _:b583719525 . . _:b583719539 "2"^^ . _:b583719524 . _:b583719519 . _:b583719538 "2"^^ . _:b583719527 . _:b42336765 "We define a function for the ELF domain, which is conserved among FANCL species (19), although dispensable in vitro for both substrate binding and catalysis of FANCD2 monoubiquitination (19\u2013>>21<<). Our finding that the ubiquitin-binding patch of the ELF domain is required for efficient FANCD2 monoubiquitination in cells suggest that ELF domain mutations would be harmful. However, there are as yet no FA patients identified with" . _:b583719549 "2"^^ . _:b583719508 . _:b583719526 . _:b583719548 "2"^^ . . _:b583719521 . . _:b42336765 . _:b583719551 "2"^^ . _:b583719520 . _:b583719550 "2"^^ . _:b583719523 . _:b42336775 "In addition to the requirement for Rad18 activity, PCNA and FANCD2 interact in cells (>>46<<). FANCL and PCNA are also reported to interact in cells, via the central (DRWD) domain of FANCL (47). Although the monoubiquitination of PCNA appears to be critical for monoubiquitination of FANCD2, the molecular and mechanistic details" . . _:b583719545 "2"^^ . _:b583719522 . _:b583719544 "2"^^ . _:b583719497 . _:b583719533 . _:b583719544 . _:b583719545 . _:b583719546 . _:b583719547 "2"^^ . _:b583719547 . _:b583719532 . _:b583719548 . _:b583719549 . _:b583719550 . _:b583719546 "2"^^ . _:b583719551 . _:b583719552 . _:b583719536 . _:b583719535 . . _:b583719537 . . _:b583719538 . _:b583719557 "2"^^ . _:b583719539 . _:b583719534 . _:b583719540 . . _:b583719541 . _:b583719542 . _:b583719556 "2"^^ . _:b583719543 . _:b583719529 . _:b42336783 . _:b42336752 "A, superposition of the ELF domain from Drosophila FANCL in blue (PDB 3K1L) (>>19<<) with the E2 Ube2L3 in yellow (PDB 1FBV) (48)." . _:b583719528 . _:b583719529 . _:b42336727 . _:b583719530 . _:b583719531 . _:b583719496 . _:b583719528 . _:b583719532 . _:b42336732 . _:b583719533 . _:b583719534 . _:b583719535 . _:b583719531 . _:b583719520 . _:b583719521 . _:b583719527 . _:b583719522 . _:b583719553 "2"^^ . _:b583719523 . _:b583719530 . _:b42336772 . _:b583719524 . _:b583719525 . _:b42336756 . _:b42336746 . _:b583719526 . _:b583719552 "2"^^ . _:b583719527 . _:b583719541 . _:b42336773 . _:b583719512 . _:b583719513 . _:b583719514 . _:b583719555 "2"^^ . _:b583719515 . _:b583719540 . _:b42336774 . _:b583719516 . _:b583719517 . _:b583719518 . _:b583719554 "2"^^ . _:b583719519 . _:b583719543 . _:b42336775 . _:b583719504 . _:b583719505 . _:b583719506 . _:b583719507 . _:b42336771 . _:b583719542 . _:b42336768 . _:b583719508 . _:b583719509 . _:b583719526 . _:b583719510 . _:b583719511 . _:b583719537 . _:b42336769 . _:b583719496 . . _:b583719497 . _:b583719498 . _:b583719499 . _:b583719536 . _:b42336770 . _:b583719500 . _:b583719501 . . _:b583719502 . _:b583719503 . _:b583719539 . _:b42336771 . _:b583719488 . _:b583719489 . _:b583719490 . _:b583719491 . _:b583719538 . _:b42336780 . _:b583719492 . _:b583719493 . . _:b583719494 . _:b583719495 . _:b583719549 . _:b42336781 . _:b583719480 . . _:b583719481 . _:b583719482 . _:b583719483 . _:b583719548 . _:b42336782 . _:b583719484 . _:b583719485 . _:b583719486 . _:b583719487 . _:b583719551 . _:b42336783 . _:b583719472 . _:b583719473 . _:b583719474 . _:b583719475 . _:b583719538 . _:b42336776 . _:b583719476 . _:b583719550 . _:b42336742 . _:b583719477 . _:b583719530 . . _:b583719473 . _:b583719478 . _:b583719479 . _:b42336773 "Rad18 does not monoubiquitinate FANCD2 (>>45<<). However, the E3 ligase activity of Rad18 is required for efficient loading of FANCD2 onto chromatin (44)." . _:b42336761 . _:b42336777 . . _:b583719464 . _:b583719545 . _:b583719534 . _:b583719465 . _:b583719466 . _:b42336744 "E2s form a catalytic intermediate with ubiquitin via a thioester between the catalytic cysteine and the C terminus of ubiquitin (>>29<<). The ELF domain does not possess a catalytic cysteine. However, an additional feature of E2s is that they can also interact non-covalently with ubiquitin (30, 31). Therefore we hypothesized that the ELF domain of FANCL might interact" . _:b583719467 . _:b583719544 . _:b42336778 . _:b583719468 . . _:b583719469 . _:b583719470 . _:b42336726 . _:b583719471 . _:b583719547 . _:b583719456 . _:b42336779 . _:b583719457 . _:b583719458 . _:b583719459 . _:b583719546 . _:b583719460 . _:b583719461 . _:b583719462 . _:b583719463 . _:b583719557 . _:b583719471 . _:b583719556 . _:b583719455 . _:b42336784 . _:b42336769 "PCNA is modified at Lys164 by the ring E3 ligase, Rad18 (>>40<<\u201342). FANCL and Rad18 are epistatic for ICL sensitivity and repair (43), and co-depletion of FANCD2 and Rad18 does not increase cellular sensitivity to cisplatin, suggesting the proteins function in the same pathway (44)." . _:b42336754 "However, ubiquitin-interacting proteins and motifs such as CUE domains, UBAs, UIMs, and MIUs often share very little sequence homology yet retain functional homology (>>33<<). We therefore wanted to determine whether the function of ubiquitin binding is conserved in vertebrate FANCL homologs. We and others had difficulties to make wild type full-length human FANCL, therefore we turned to the Xenopus system." . _:b583719553 . _:b42336785 . . _:b42336756 "Several E2-RING E3 ligase interactions are enhanced by the presence of the ubiquitin thioester bound on the E2 (34, >>35<<). Therefore another explanation is that FANCL interaction with ubiquitin enhances the recognition of ubiquitin-charged Ube2T (Ube2T\u223CUb)." . _:b583719552 . _:b42336786 . _:b42336781 "We expressed and purified the protein as previously described (>>24<<). Briefly, Hi5 cells were infected with baculovirus, and the protein was purified on FLAG resin before elution with 3\u00D7 FLAG peptide and stored with 5% glycerol at \u221280 \u00B0C." . _:b42336721 . _:b42336787 . _:b583719555 . _:b583719554 . . . _:b42336725 "Biallelic mutations in any of 16 currently identified FA genes results in failure to repair DNA interstrand crosslinks (ICL), which can occur during replicative stress and upon encountering endogenous genotoxins (3\u2013>>9<<)." . . . _:b583719529 . . . . _:b583719524 . . . _:b583719456 . . . _:b42336768 "ICL repair is a complex and multistep process, that also requires components of the translesion synthesis (TLS) pathway (9, 38, >>39<<). In common with the FA pathway, the TLS pathway is also regulated by a site-specific monoubiquitination event. PCNA is modified at Lys164 by the ring E3 ligase, Rad18 (40\u201342). FANCL and Rad18 are epistatic for ICL sensitivity and repair" . . _:b583719545 . . _:b42336760 . _:b42336721 _:b42336736 . _:b42336721 _:b42336737 . _:b42336721 _:b42336738 . _:b42336721 _:b42336739 . _:b42336721 _:b42336740 . _:b42336721 _:b42336741 . _:b583719485 . _:b42336731 . _:b583719515 . _:b42336762 . . _:b42336723 . _:b583719531 . _:b42336750 "of FANCL are not amenable to large scale soluble expression (20, 23); therefore, we used the more soluble invertebrate ELF domain from Drosophila, which shares \u223C65% sequence similarity (19% identity) with the human ELF domain (>>19<<). First, we determined the solution structure of the ELF domain." . _:b583719547 . _:b583719470 . . _:b42336721 _:b42336722 . _:b42336749 "The mammalian and vertebrate homologues of FANCL are not amenable to large scale soluble expression (20, >>23<<); therefore, we used the more soluble invertebrate ELF domain from Drosophila, which shares \u223C65% sequence similarity (19% identity) with the human ELF domain (19)." . . _:b42336721 _:b42336723 . _:b42336721 _:b42336724 . . _:b42336721 _:b42336725 . . _:b42336721 _:b42336726 . _:b42336721 _:b42336727 . _:b42336721 _:b42336728 . _:b42336721 _:b42336729 . _:b42336721 _:b42336730 . _:b42336769 . _:b42336721 _:b42336731 . _:b42336721 _:b42336732 . _:b42336721 _:b42336733 . _:b42336759 . _:b42336721 _:b42336734 . _:b42336721 _:b42336735 . _:b583719493 . _:b583719512 . _:b42336724 "Biallelic mutations in any of 16 currently identified FA genes results in failure to repair DNA interstrand crosslinks (ICL), which can occur during replicative stress and upon encountering endogenous genotoxins (>>3<<\u20139)." . _:b42336745 . _:b42336742 _:b42336760 . . . _:b42336742 _:b42336756 . _:b42336760 "In a previous study based on this prediction, mutation of the predicted WD40 repeats, including a large part of the ELF domain, was found to disrupt assembly of the FA core complex (>>16<<). Since the core complex is required for efficient FANCL-catalyzed FANCD2 monoubiquitination in cells, we next assessed whether the defective FANCI/FANCD2 monoubiquitination in TAP-FANCL (L7A, D78R, L79A) expressing cells was due to" . _:b42336742 _:b42336757 . . . _:b42336742 _:b42336758 . _:b42336742 _:b42336759 . _:b42336747 . _:b42336742 _:b42336752 . _:b42336742 _:b42336753 . _:b42336742 _:b42336754 . _:b42336742 _:b42336755 . _:b42336742 _:b42336748 . _:b42336786 "DT40 cell transfections, DT40 subfractionation, Superpose 6 gel size chromatography, and FANCD2 immunoblot analyses were described previously (>>27<<). To assess TAP-FANCL interaction with ubiquitin, fancl\u2212/\u2212 cells expressing either TAP-FANCL or TAP-FANCL (L7A, D78R, L79A) were lysed in TAP-buffer (50 mm Tris, pH 8.0, 200 mm NaCl, 0.5 mm EDTA, 0.1% Nonidet P-40, and protease inhibitor" . _:b42336742 _:b42336749 . _:b583719552 . _:b42336742 _:b42336750 . _:b583719553 . _:b42336742 _:b42336751 . _:b583719554 . _:b583719475 . _:b42336742 _:b42336744 . _:b583719555 . _:b42336742 _:b42336745 . _:b583719556 . _:b42336742 _:b42336746 . _:b583719557 . _:b42336742 _:b42336747 . _:b42336742 _:b42336743 . . _:b583719461 . _:b583719522 . _:b42336778 "Alternatively, given that monoubiquitination of PCNA is required for accumulation of FANCA on chromatin (>>45<<), monoubiquitinated PCNA could aid in recruitment of the core complex to sites of DNA damage and/or activation of FANCL." . _:b42336787 . _:b42336767 . _:b42336721 "introduction" . _:b583719503 . _:b583719460 . . _:b42336741 "However, the ELF domain has no known function and makes no contacts with the other domains of FANCL (>>19<<). The ELF domain is found in all FANCL homologues, and is conserved across species. Therefore, we sought to establish the function of the ELF domain. We report here a previously undetected non-covalent interaction between the ELF domain" . . _:b42336744 . _:b42336753 "A, superposition of the ELF domain from Drosophila FANCL in blue (PDB 3K1L) (19) with the E2 Ube2L3 in yellow (PDB 1FBV) (>>48<<). E2 protein-protein interaction surfaces indicated, as is the position of the catalytic cysteine of Ube2L3. B, assignment of the Drosophila ELF domain 1H-15N HSQC. The cross-peaks in the 1H-15N HSQC were assigned to residues in the" . . . _:b583719489 . . _:b42336761 "discussion" . . _:b42336726 "A key step in DNA ICL repair is the site-specific monoubiquitination of FANCD2 at Lys561 (>>10<<), which leads to the recruitment of downstream repair factors. This monoubiquitination event is carried out by FANCL, the RING E3 ligase subunit of the FA core complex (11, 12)." . _:b42336743 "The ELF domain of FANCL shares significant structural homology with E2-conjugating enzymes (>>19<<). E2s form a catalytic intermediate with ubiquitin via a thioester between the catalytic cysteine and the C terminus of ubiquitin (29)." . _:b583719479 . . _:b583719516 . _:b42336753 . . _:b583719481 . _:b42336742 "results" . _:b42336755 . _:b42336761 _:b42336762 . _:b583719539 . . _:b42336761 _:b42336763 . . _:b42336761 _:b42336764 . _:b42336761 _:b42336765 . _:b42336761 _:b42336766 . _:b42336761 _:b42336767 . . _:b583719533 . . _:b583719468 . _:b583719518 . . . . . _:b42336739 "The DRWD domain harbors the substrate binding site (19, >>20<<, 23). However, the ELF domain has no known function and makes no contacts with the other domains of FANCL (19)." . _:b583719498 . . _:b42336730 . _:b583719459 . _:b583719551 . . _:b42336728 . _:b42336779 . _:b583719474 . _:b583719469 . _:b583719517 . _:b583719525 . . _:b42336748 "The mammalian and vertebrate homologues of FANCL are not amenable to large scale soluble expression (>>20<<, 23); therefore, we used the more soluble invertebrate ELF domain from Drosophila, which shares \u223C65% sequence similarity (19% identity) with the human ELF domain (19)." . _:b42336772 . . _:b42336729 "event is carried out by FANCL, the RING E3 ligase subunit of the FA core complex (11, 12). In vertebrates, the FA core complex comprises 9 proteins: FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FAAP100, FAAP20, and FANCL, reviewed in Ref. >>13<<. Recent studies have shown that the core complex members FANCB, and FAAP100 are required for full ubiquitin ligase activity in cells (14, 15)." . _:b42336774 . _:b583719462 . _:b583719494 . _:b583719514 . . "10.1074%2Fjbc.M115.675835" . . _:b42336777 "We propose that the ELF domain of FANCL interacts with monoubiquitinated PCNA, and that may act as a trigger for FANCD2 monoubiquitination, as observed by Geng et al. (>>47<<). Potentially, monoubiquitinated PCNA reinforces the interaction between FANCD2 and FANCI. Alternatively, given that monoubiquitination of PCNA is required for accumulation of FANCA on chromatin (45), monoubiquitinated PCNA could aid in" . . . _:b42336737 "The RING domain serves as an E2-conjugating enzyme recruitment module, being necessary and sufficient for the interaction with Ube2T, the E2 for the FA pathway (20\u2013>>22<<). The DRWD domain harbors the substrate binding site (19, 20, 23). However, the ELF domain has no known function and makes no contacts with the other domains of FANCL (19). The ELF domain is found in all FANCL homologues, and is conserved" . _:b583719507 . _:b42336766 "ICL repair is a complex and multistep process, that also requires components of the translesion synthesis (TLS) pathway (>>9<<, 38, 39). In common with the FA pathway, the TLS pathway is also regulated by a site-specific monoubiquitination event. PCNA is modified at Lys164 by the ring E3 ligase, Rad18 (40\u201342). FANCL and Rad18 are epistatic for ICL sensitivity and" . . _:b42336782 . _:b583719528 . _:b583719482 . _:b42336776 "FANCL and PCNA are also reported to interact in cells, via the central (DRWD) domain of FANCL (>>47<<). Although the monoubiquitination of PCNA appears to be critical for monoubiquitination of FANCD2, the molecular and mechanistic details of this interplay are poorly understood. We propose that the ELF domain of FANCL interacts with" . _:b42336767 "ICL repair is a complex and multistep process, that also requires components of the translesion synthesis (TLS) pathway (9, >>38<<, 39). In common with the FA pathway, the TLS pathway is also regulated by a site-specific monoubiquitination event. PCNA is modified at Lys164 by the ring E3 ligase, Rad18 (40\u201342). FANCL and Rad18 are epistatic for ICL sensitivity and" . . _:b583719455 . _:b583719476 . _:b42336746 "However, an additional feature of E2s is that they can also interact non-covalently with ubiquitin (30, >>31<<). Therefore we hypothesized that the ELF domain of FANCL might interact with ubiquitin in a similar manner. To test this hypothesis, we performed a pull-down binding assay using 6\u00D7 His-tagged ubiquitin as bait (Fig. 1A). His-ubiquitin" . _:b42336764 "We define a function for the ELF domain, which is conserved among FANCL species (19), although dispensable in vitro for both substrate binding and catalysis of FANCD2 monoubiquitination (>>19<<\u201321). Our finding that the ubiquitin-binding patch of the ELF domain is required for efficient FANCD2 monoubiquitination in cells suggest that ELF domain mutations would be harmful. However, there are as yet no FA patients identified with" . . . _:b583719505 . _:b583719537 . _:b42336784 "All NMR data processing utilized NMRPipe (25) and analyzed using the CCPN analysis package (>>26<<). Most resonances were successfully assigned manually without ambiguity. Backbone chemical shift assignments were achieved for the ELF domain using 1H-15N HSQC, CBCA(CO)NH, CBCANH, 15N-NOESY-HSQC, and 15N-TOCSY-HSQC experiments." . . _:b583719510 . _:b42336755 "Several E2-RING E3 ligase interactions are enhanced by the presence of the ubiquitin thioester bound on the E2 (>>34<<, 35). Therefore another explanation is that FANCL interaction with ubiquitin enhances the recognition of ubiquitin-charged Ube2T (Ube2T\u223CUb)." . . _:b42336758 . . _:b583719556 . . . _:b42336757 . _:b583719542 . _:b583719554 . _:b42336776 . . _:b42336777 . . _:b42336722 "Fanconi Anemia (FA)3 is an autosomal recessive or X-linked inherited childhood disorder, characterized by bone marrow failure and a high incidence of cancer (>>1<<, 2). Biallelic mutations in any of 16 currently identified FA genes results in failure to repair DNA interstrand crosslinks (ICL), which can occur during replicative stress and upon encountering endogenous genotoxins (3\u20139)." . _:b583719511 . . _:b583719506 . _:b42336749 . _:b583719540 . . _:b42336772 "FANCL and Rad18 are epistatic for ICL sensitivity and repair (43), and co-depletion of FANCD2 and Rad18 does not increase cellular sensitivity to cisplatin, suggesting the proteins function in the same pathway (>>44<<). Rad18 does not monoubiquitinate FANCD2 (45). However, the E3 ligase activity of Rad18 is required for efficient loading of FANCD2 onto chromatin (44). In addition to the requirement for Rad18 activity, PCNA and FANCD2 interact in cells" . _:b583719549 . _:b42336729 . . _:b583719484 . . . . _:b42336784 . . . _:b583719483 . _:b42336771 "FANCL and Rad18 are epistatic for ICL sensitivity and repair (>>43<<), and co-depletion of FANCD2 and Rad18 does not increase cellular sensitivity to cisplatin, suggesting the proteins function in the same pathway (44)." . . . . _:b42336740 . . . . . . . _:b42336737 . _:b42336734 . . _:b583719491 . . _:b583719543 . _:b42336763 "We define a function for the ELF domain, which is conserved among FANCL species (>>19<<), although dispensable in vitro for both substrate binding and catalysis of FANCD2 monoubiquitination (19\u201321)." . . _:b42336780 . _:b583719480 . _:b583719521 . _:b583719520 . . . _:b42336774 "However, the E3 ligase activity of Rad18 is required for efficient loading of FANCD2 onto chromatin (>>44<<). In addition to the requirement for Rad18 activity, PCNA and FANCD2 interact in cells (46). FANCL and PCNA are also reported to interact in cells, via the central (DRWD) domain of FANCL (47). Although the monoubiquitination of PCNA" . _:b42336745 "However, an additional feature of E2s is that they can also interact non-covalently with ubiquitin (>>30<<, 31). Therefore we hypothesized that the ELF domain of FANCL might interact with ubiquitin in a similar manner. To test this hypothesis, we performed a pull-down binding assay using 6\u00D7 His-tagged ubiquitin as bait (Fig. 1A). His-ubiquitin" . _:b583719550 . . _:b583719457 . _:b583719548 . _:b583719465 . _:b583719467 . . _:b583719500 . _:b42336724 . _:b42336731 "Recent studies have shown that the core complex members FANCB, and FAAP100 are required for full ubiquitin ligase activity in cells (14, >>15<<). Previous work has also suggested that FANCL is required for core complex assembly (16). Intriguingly, while FANCL as the functional ligase subunit is conserved in invertebrates, there are no identifiable homologs of FANCB or FAAP100 in" . _:b42336785 . _:b42336725 . _:b42336786 . . _:b42336726 . . _:b42336722 . _:b42336727 . _:b42336766 . . . _:b42336721 . _:b42336722 . . _:b42336723 . _:b42336732 . _:b42336750 . _:b42336733 . _:b42336734 . _:b42336769 . _:b42336735 . . _:b42336770 "PCNA is modified at Lys164 by the ring E3 ligase, Rad18 (40\u2013>>42<<). FANCL and Rad18 are epistatic for ICL sensitivity and repair (43), and co-depletion of FANCD2 and Rad18 does not increase cellular sensitivity to cisplatin, suggesting the proteins function in the same pathway (44)." . _:b42336728 . _:b42336738 . _:b42336729 . . _:b42336730 . . _:b42336731 . _:b42336743 . _:b583719466 . _:b42336740 . _:b42336779 _:b42336780 . _:b42336779 _:b42336781 . _:b42336779 _:b42336782 . _:b42336741 . _:b42336779 _:b42336783 . _:b42336754 . _:b42336741 . _:b42336742 . _:b42336743 . _:b583719513 . _:b42336763 . _:b42336736 . _:b42336778 . _:b42336737 . _:b583719455 "5"^^ . . _:b42336779 _:b42336784 . _:b42336779 _:b42336785 . _:b42336779 _:b42336786 . _:b42336728 "This monoubiquitination event is carried out by FANCL, the RING E3 ligase subunit of the FA core complex (11, >>12<<). In vertebrates, the FA core complex comprises 9 proteins: FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FAAP100, FAAP20, and FANCL, reviewed in Ref. 13. Recent studies have shown that the core complex members FANCB, and FAAP100 are required" . _:b42336738 . _:b42336779 _:b42336787 . _:b42336739 . _:b42336748 . _:b583719555 . . . _:b42336749 . . _:b583719532 . _:b42336750 . _:b42336764 . _:b583719461 "4"^^ . _:b42336751 . _:b583719499 . _:b583719460 "4"^^ . . _:b42336727 "This monoubiquitination event is carried out by FANCL, the RING E3 ligase subunit of the FA core complex (>>11<<, 12). In vertebrates, the FA core complex comprises 9 proteins: FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FAAP100, FAAP20, and FANCL, reviewed in Ref. 13. Recent studies have shown that the core complex members FANCB, and FAAP100 are" . _:b42336744 . _:b583719463 "3"^^ . _:b583719464 . _:b42336745 . _:b42336735 "The crystal structure of full-length FANCL revealed 3 domains (>>19<<), an N-terminal E2-like fold (ELF) domain, a central double RWD (DRWD) domain, and a C-terminal RING domain." . _:b583719523 . _:b583719462 "4"^^ . _:b42336746 . _:b42336785 "FANCL-deficient DT40 cells (fancl\u2212/\u2212) and fancl\u2212/\u2212 complemented with TAP-tagged wild-type FANCL (TAP-FANCL) were described recently (>>27<<). Point mutations L7A, D78A, D78R, L79A, and V80A were generated by site-directed muagenesis (QuickChange II XL site-directed mutagenesis kit; Stratagene)." . . _:b42336752 . _:b583719457 "4"^^ . _:b42336747 . _:b583719456 "5"^^ . _:b42336756 . _:b583719459 "4"^^ . _:b42336757 . . _:b583719458 "4"^^ . _:b42336758 . _:b583719469 "3"^^ . _:b42336759 . _:b42336773 . _:b583719468 "3"^^ . _:b42336752 . _:b583719471 "3"^^ . _:b42336753 . _:b583719470 "3"^^ . _:b42336754 . _:b583719465 "3"^^ . _:b42336755 . _:b583719464 "3"^^ . _:b42336764 . _:b583719458 . _:b583719467 "3"^^ . _:b42336780 "Drosophila melanogaster FANCL constructs and DRWD-RING (105\u2013431) were expressed and purified as described previously (>>19<<)." . _:b42336765 . _:b42336738 "The DRWD domain harbors the substrate binding site (>>19<<, 20, 23). However, the ELF domain has no known function and makes no contacts with the other domains of FANCL (19)." . _:b583719455 . _:b583719466 "3"^^ . . . _:b42336761 _:b42336768 . . _:b42336766 . _:b42336762 "This study describes a hitherto unknown non-covalent interaction between FANCL and ubiquitin, with an affinity commonly observed in ubiquitin-protein interactions (>>36<<), that is required for efficient FANCD2 monoubiquitination. The interaction between the ELF domain of FANCL and ubiquitin is distinct from the surfaces in E2 proteins commonly used for non-covalent ubiquitin binding." . _:b42336761 _:b42336769 . _:b42336761 _:b42336770 . . _:b42336725 . _:b42336761 _:b42336771 . _:b583719477 "3"^^ . _:b42336761 _:b42336772 . _:b42336761 _:b42336773 . _:b42336767 . _:b583719535 . _:b42336761 _:b42336774 . _:b42336761 _:b42336775 . _:b583719476 "3"^^ . _:b42336761 _:b42336776 . _:b583719504 . _:b42336760 . _:b42336757 "Since Ube2T auto-monoubiquitinates (>>22<<), and FANCL has been shown to stimulate this (22), we performed a thioester discharge assay to test whether the ELF-Ubiquitin interaction is important for this." . _:b42336761 _:b42336777 . _:b42336761 _:b42336778 . _:b583719487 . _:b583719479 "3"^^ . . _:b42336761 . _:b583719478 "3"^^ . _:b42336762 . _:b583719473 "3"^^ . _:b42336763 . _:b583719472 "3"^^ . _:b583719461 . _:b583719546 . _:b583719475 "3"^^ . _:b583719460 . _:b42336733 "Indeed the only additional core complex component found in an invertebrate is FANCE in Dictyostelium (>>17<<, 18)." . _:b583719474 "3"^^ . _:b583719463 . _:b583719485 "2"^^ . _:b583719462 . . _:b583719541 . _:b583719484 "2"^^ . "PMC0" . _:b583719457 . _:b42336734 "Indeed the only additional core complex component found in an invertebrate is FANCE in Dictyostelium (17, >>18<<)." . _:b583719487 "2"^^ . _:b583719456 . . _:b583719486 "2"^^ . _:b583719459 . _:b583719463 . _:b583719458 . _:b583719481 "3"^^ . _:b583719502 . _:b42336735 . _:b583719469 . _:b583719480 "3"^^ . _:b583719501 . _:b583719483 "2"^^ . _:b583719468 . _:b42336770 . . _:b583719471 . _:b583719482 "3"^^ . . _:b583719493 "2"^^ . _:b583719492 . _:b583719470 . . . _:b583719492 "2"^^ . _:b583719465 . . _:b583719495 "2"^^ . _:b583719464 . _:b583719494 "2"^^ . _:b583719467 . _:b583719489 "2"^^ . _:b583719466 . . _:b583719488 "2"^^ . _:b583719477 . _:b583719491 "2"^^ . _:b583719476 . _:b583719490 "2"^^ . _:b583719479 . _:b42336747 "E2s bind ubiquitin non-covalently via a backside interaction that involves residues from the loop connecting strands \u03B22 and \u03B23 (>>30<<) (Fig. 2A). The dissociation constant between ubiquitin and the ELF domain suggests that crystallization of the complex would prove challenging. Indeed, despite extensive efforts, we were unable to obtain high-resolution diffracting" . _:b583719501 "2"^^ . _:b583719478 . . _:b583719500 "2"^^ . _:b42336779 "experimental procedures" . _:b583719473 . _:b583719472 . _:b583719503 "2"^^ . _:b583719472 . . _:b583719502 "2"^^ . . _:b583719475 . _:b42336783 "All NMR data processing utilized NMRPipe (>>25<<) and analyzed using the CCPN analysis package (26). Most resonances were successfully assigned manually without ambiguity." . _:b583719536 . _:b583719478 . _:b583719497 "2"^^ . _:b583719474 . _:b42336759 "FANCL was originally predicted to adopt a WD40-propeller fold in place of the ELF-DRWD domains (>>12<<). In a previous study based on this prediction, mutation of the predicted WD40 repeats, including a large part of the ELF domain, was found to disrupt assembly of the FA core complex (16)." . _:b42336724 . . _:b583719496 "2"^^ . _:b42336723 "Fanconi Anemia (FA)3 is an autosomal recessive or X-linked inherited childhood disorder, characterized by bone marrow failure and a high incidence of cancer (1, >>2<<). Biallelic mutations in any of 16 currently identified FA genes results in failure to repair DNA interstrand crosslinks (ICL), which can occur during replicative stress and upon encountering endogenous genotoxins (3\u20139)." . _:b583719485 . _:b42336736 "The RING domain serves as an E2-conjugating enzyme recruitment module, being necessary and sufficient for the interaction with Ube2T, the E2 for the FA pathway (>>20<<\u201322). The DRWD domain harbors the substrate binding site (19, 20, 23). However, the ELF domain has no known function and makes no contacts with the other domains of FANCL (19). The ELF domain is found in all FANCL homologues, and is" . _:b42336764 . _:b42336751 "4, A and B) (>>32<<). These results reveal a novel interaction surface on the ELF domain." . _:b583719499 "2"^^ . _:b583719484 . _:b42336781 . _:b583719498 "2"^^ . _:b583719487 . _:b583719509 "2"^^ . _:b583719486 . _:b583719557 . _:b583719508 "2"^^ . . _:b583719481 . _:b583719511 "2"^^ . _:b583719480 . . _:b42336732 "Previous work has also suggested that FANCL is required for core complex assembly (>>16<<). Intriguingly, while FANCL as the functional ligase subunit is conserved in invertebrates, there are no identifiable homologs of FANCB or FAAP100 in lower eukaryotes. Indeed the only additional core complex component found in an" . _:b583719510 "2"^^ . _:b583719483 . . _:b42336724 . _:b583719505 "2"^^ . _:b42336787 "Charged Ube2T was prepared by adapting the method described in Ref. >>28<<. Human His-Ube2T C24S and tag-free ubiquitin G76C were purified to homogeneity as before, albeit retaining the His-tag on Ube2T." . _:b583719482 . _:b583719504 "2"^^ . _:b583719493 . . _:b42336730 "Recent studies have shown that the core complex members FANCB, and FAAP100 are required for full ubiquitin ligase activity in cells (>>14<<, 15). Previous work has also suggested that FANCL is required for core complex assembly (16). Intriguingly, while FANCL as the functional ligase subunit is conserved in invertebrates, there are no identifiable homologs of FANCB or FAAP100" . . _:b583719507 "2"^^ . _:b583719492 . _:b583719506 "2"^^ . . _:b583719495 . _:b583719517 "2"^^ . _:b583719494 . _:b583719516 "2"^^ . _:b583719489 . _:b583719519 "2"^^ . _:b583719488 . . _:b42336733 . _:b42336740 "The DRWD domain harbors the substrate binding site (19, 20, >>23<<). However, the ELF domain has no known function and makes no contacts with the other domains of FANCL (19)." . _:b583719495 . . _:b583719518 "2"^^ . _:b583719490 . _:b583719491 . . . _:b583719513 "2"^^ . _:b583719490 . . _:b583719512 "2"^^ . _:b583719501 . _:b42336748 . . _:b583719515 "2"^^ . _:b583719500 . _:b583719514 "2"^^ . _:b583719503 . _:b583719525 "2"^^ . _:b583719502 . . _:b583719524 "2"^^ . _:b583719497 . _:b42336736 . _:b42336751 . _:b42336768 . _:b583719527 "2"^^ . _:b583719496 . _:b42336739 . . _:b583719526 "2"^^ . _:b583719499 . _:b42336782 "FANCL was expressed and purified as previously described (>>21<<)." . _:b583719509 . _:b583719521 "2"^^ . _:b583719498 . . . _:b42336775 . _:b583719520 "2"^^ . _:b583719509 . _:b583719523 "2"^^ . _:b583719508 . _:b583719553 . _:b583719522 "2"^^ . _:b583719511 . _:b583719533 "2"^^ . . _:b583719510 . _:b583719532 "2"^^ . _:b583719505 . _:b583719535 "2"^^ . _:b583719504 . _:b583719534 "2"^^ . _:b583719507 . _:b583719488 .