@prefix rdf: . @prefix ns1: . rdf:type ns1:RelevantPaper , ns1:ReferencePaper , ns1:CitationPaper . @prefix rdfs: . rdfs:seeAlso , , , . @prefix bibo: . bibo:cites , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ; ns1:cocitationWith , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , ; ns1:hasRelevantBibliographicResourceOf _:b583719544 , _:b583719545 , _:b583719546 , _:b583719547 , _:b583719548 , _:b583719549 , _:b583719550 , _:b583719551 , _:b583719536 , _:b583719537 , _:b583719538 , _:b583719539 , _:b583719540 , _:b583719541 , _:b583719542 , _:b583719543 , _:b583719528 , _:b583719529 , _:b583719530 , _:b583719531 , _:b583719532 , _:b583719533 , _:b583719534 , _:b583719535 , _:b583719520 , _:b583719521 , _:b583719522 , _:b583719523 , _:b583719524 , _:b583719525 , _:b583719526 , _:b583719527 , _:b583719512 , _:b583719513 , _:b583719514 , _:b583719515 , _:b583719516 , _:b583719517 , _:b583719518 , _:b583719519 , _:b583719504 , _:b583719505 , _:b583719506 , _:b583719507 , _:b583719508 , _:b583719509 , _:b583719510 , _:b583719511 , _:b583719496 , _:b583719497 , _:b583719498 , _:b583719499 , _:b583719500 , _:b583719501 , _:b583719502 , _:b583719503 , _:b583719488 , _:b583719489 , _:b583719490 , _:b583719491 , _:b583719492 , _:b583719493 , _:b583719494 , _:b583719495 , _:b583719480 , _:b583719481 , _:b583719482 , _:b583719483 , _:b583719484 , _:b583719485 , _:b583719486 , _:b583719487 , _:b583719472 , _:b583719473 , _:b583719474 , _:b583719475 , _:b583719476 , _:b583719477 , _:b583719478 , _:b583719479 , _:b583719464 , _:b583719465 , _:b583719466 , _:b583719467 , _:b583719468 , _:b583719469 , _:b583719470 , _:b583719471 , _:b583719456 , _:b583719457 , _:b583719458 , _:b583719459 , _:b583719460 , _:b583719461 , _:b583719462 , _:b583719463 , _:b583719455 , _:b583719552 , _:b583719553 , _:b583719554 , _:b583719555 , _:b583719556 , _:b583719557 ; ns1:pmcid "PMC0" ; bibo:doi "10.1074%2Fjbc.M115.675835" . @prefix ns4: . ns4:contains _:b42336742 , _:b42336761 , _:b42336721 , _:b42336779 . _:b42336721 rdf:type ns4:Section . @prefix dc: . _:b42336721 dc:title "introduction" ; ns4:contains _:b42336736 , _:b42336737 , _:b42336738 , _:b42336739 , _:b42336740 , _:b42336741 , _:b42336722 , _:b42336723 , _:b42336724 , _:b42336725 , _:b42336726 , _:b42336727 , _:b42336728 , _:b42336729 , _:b42336730 , _:b42336731 , _:b42336732 , _:b42336733 , _:b42336734 , _:b42336735 . _:b42336722 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336723 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336724 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions , . _:b42336725 rdf:type ns1:Context ; rdf:value "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<<)." ; ns1:mentions . _:b42336726 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336727 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336728 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336729 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336730 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336731 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336732 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336733 rdf:type ns1:Context ; rdf:value "Indeed the only additional core complex component found in an invertebrate is FANCE in Dictyostelium (>>17<<, 18)." ; ns1:mentions . _:b42336734 rdf:type ns1:Context ; rdf:value "Indeed the only additional core complex component found in an invertebrate is FANCE in Dictyostelium (17, >>18<<)." ; ns1:mentions . _:b42336735 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336736 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions , . _:b42336737 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336738 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336739 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336740 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336741 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336742 rdf:type ns4:Section ; dc:title "results" ; ns4:contains _:b42336760 , _:b42336756 , _:b42336757 , _:b42336758 , _:b42336759 , _:b42336752 , _:b42336753 , _:b42336754 , _:b42336755 , _:b42336748 , _:b42336749 , _:b42336750 , _:b42336751 , _:b42336744 , _:b42336745 , _:b42336746 , _:b42336747 , _:b42336743 . _:b42336743 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336744 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336745 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336746 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336747 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336748 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336749 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336750 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336751 rdf:type ns1:Context ; rdf:value "4, A and B) (>>32<<). These results reveal a novel interaction surface on the ELF domain." ; ns1:mentions . _:b42336752 rdf:type ns1:Context ; rdf:value "A, superposition of the ELF domain from Drosophila FANCL in blue (PDB 3K1L) (>>19<<) with the E2 Ube2L3 in yellow (PDB 1FBV) (48)." ; ns1:mentions . _:b42336753 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336754 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336755 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336756 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336757 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336758 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336759 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336760 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336761 rdf:type ns4:Section ; dc:title "discussion" ; ns4:contains _:b42336762 , _:b42336763 , _:b42336764 , _:b42336765 , _:b42336766 , _:b42336767 , _:b42336768 , _:b42336769 , _:b42336770 , _:b42336771 , _:b42336772 , _:b42336773 , _:b42336774 , _:b42336775 , _:b42336776 , _:b42336777 , _:b42336778 . _:b42336762 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336763 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336764 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions , . _:b42336765 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336766 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336767 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336768 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336769 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions , . _:b42336770 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336771 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336772 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336773 rdf:type ns1:Context ; rdf:value "Rad18 does not monoubiquitinate FANCD2 (>>45<<). However, the E3 ligase activity of Rad18 is required for efficient loading of FANCD2 onto chromatin (44)." ; ns1:mentions . _:b42336774 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336775 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336776 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336777 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336778 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336779 rdf:type ns4:Section ; dc:title "experimental procedures" ; ns4:contains _:b42336780 , _:b42336781 , _:b42336782 , _:b42336783 , _:b42336784 , _:b42336785 , _:b42336786 , _:b42336787 . _:b42336780 rdf:type ns1:Context ; rdf:value "Drosophila melanogaster FANCL constructs and DRWD-RING (105\u2013431) were expressed and purified as described previously (>>19<<)." ; ns1:mentions . _:b42336781 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336782 rdf:type ns1:Context ; rdf:value "FANCL was expressed and purified as previously described (>>21<<)." ; ns1:mentions . _:b42336783 rdf:type ns1:Context ; rdf:value "All NMR data processing utilized NMRPipe (>>25<<) and analyzed using the CCPN analysis package (26). Most resonances were successfully assigned manually without ambiguity." ; ns1:mentions . _:b42336784 rdf:type ns1:Context ; rdf:value "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." ; ns1:mentions . _:b42336785 rdf:type ns1:Context ; rdf:value "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)." ; ns1:mentions . _:b42336786 rdf:type ns1:Context ; rdf:value "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" ; ns1:mentions . _:b42336787 rdf:type ns1:Context ; rdf:value "Charged Ube2T was prepared by adapting the method described in Ref. >>28<<. 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