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2014 Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL Joseph McClanaghan University of Rhode Island, [email protected] Creative Commons License

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Recommended Citation McClanaghan, Joseph, "Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL proteins" (2014). Senior Honors Projects. Paper 367. http://digitalcommons.uri.edu/srhonorsprog/367http://digitalcommons.uri.edu/srhonorsprog/367

This Article is brought to you for free and open access by the Honors Program at the University of Rhode Island at DigitalCommons@URI. It has been accepted for inclusion in Senior Honors Projects by an authorized administrator of DigitalCommons@URI. For more information, please contact [email protected]. Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL proteins Joseph D. McClanaghan Karissa L. Paquin, Paul A. Azzinaro; Faculty Sponsor Dr. Niall G. Howlett Department of Cell and Molecular Biology, University of Rhode Island, Kingston RI 02881 Background A pCMV- S1 FLAG/HA pLenti6.2- HPV16 E7 HA-FANCL (FA) is a rare X-linked and autosomal disease CUE COS7 + characterized by bone marrow failure, congenital defects, and S1/S2 DNA (ug) ND 1 2 6 8 increased cancer susceptibility. FA is caused by mutations in S2 any 1 of 16 . The products of these genes work S2/S3 together in the FA-BRCA DNA damage response pathway to S3 repair DNA interstrand crosslinks (ICLs). The FA-BRCA FANCL HBD pathway is a critical tumor suppressor pathway. α-HA.11 Chromodomain S4

HPV16 E7 B

V5 FANCD2 α-Tubulin Tubulin V5 FANCD2 S1 1 2 3 4 5 V5 FANCD2 S2 Figure 6. Expression of pLenti6.2 HA-FANCL. COS7 cells were V5 FANCD2 S3 transfected with pLenti6.2-HA-FANCL, a FLAG/HA HPV16 V5 FANCD2 S4 control, or no DNA (ND). Cells were harvested and lysed in 2% Figure 3. Generation of overlapping fragments of FANCD2. SDS lysis buffer and analyzed via Western blotting using anti- Four FANCD2 fragments were generated, each with a N- HA and anti-tubulin antibodies. terminal V5 epitope tag. The four fragments are based on the Input IP: V5 Agarose four solenoids of FANCD2, designated S1-S4.

COS7 + D2-S1 D2-S2 D2-S3 D2-S4 DNA (ug) ND 1 2 4 8 1 2 1 2

D2S3 COS7 +

α-V5 α-D2 E35 FAND2-Ub D2S1 FAND2 Figure 1. Schematic model of the FA-BRCA pathway. Upon ICL D2S2 formation, the FA core complex monoubiquitinates FANCD2 D2S4 and FANCI. Upon monoubiquitination FANCD2 and FANCI α-Tubulin Tubulin α-HA.11 localize to sites of DNA damage where they interact with FANCL known DNA repair proteins. 1 2 3 4 5 6 7 8 9 Figure 4. Expression of pcDNA3.1-V5-FANCD2 Fragments. 1 2 3 4 5 6 7 8 COS7 cells were transfected with pcDNA3.1-V5-FANCD2 Figure 7. Co-Immunoprecipitation of V5-FANCD2 and HA- A B Substrate Ub D2 Ub fragments or no DNA (ND). Cells were harvested and lysed in FANCL. A co-IP was performed to determine if FANCD2 and 2% SDS lysis buffer, and analyzed via Western blotting using FANCL interact. COS7 cells were transfected with no DNA F-Box UBC UBE2T E Protein L anti-V5 and anti-tubulin antibodies. (ND; lanes 3 and 7), pcDNA3.1-V5-FANCD2 (V5-D2; lanes 1 and RBX1 pCMV- 5), pLenti6.2-HA-FANCL (HA-L; lanes 2 and 6), or both (V5-D2 + Skp1 C FLAG/HA pEAK8- HA-L; lanes 4 and 8). Samples were lysed in low stringency Cullin A COS7 + HPV16 E7 FLAG-FANCE buffer and immune complexes isolated with anti-V5 agarose. DNA (ug) ND 1 1 2 3 Samples were eluted and analyzed using Western blotting with anti-HA and anti-FANCD2 antibodies. SCF E2/E3 FA-BRCA E2/E3 Complex Complex α-FLAG FANCE Conclusions We have successfully generated mammalian expression Figure 2. Comparison of SCF E2/E3 and the FA-BRCA E2/E3 constructs for four overlapping V5-tagged FANCD2 fragments ligase complexes. The SCF (for Skp1-Cul1-F-box α-Tubulin Tubulin and HA-tagged FANCL. We have also successfully optimized protein) E3 ubiquitin ligase (A) is a large complex made up of conditions for their ectopic expression in COS7 cells and also the RBX1 E3 ubiquitin ligase, UBC E2 ubiquitin-conjugating 1 2 3 4 5 optimized the FLAG-tagged FANCE construct. In addition, we enzyme, and the F-box protein which recognizes the substrate. Figure 5. Expression of FLAG-FANCE. A FLAG-tagged FANCE have begun to perform co-IP experiments to determine the Here, we propose a similar model for the ubiquitination of construct was also obtained. COS7 cells were transfected with specific regions of FANCD2 to which FANCL and FANCE bind. FANCD2 by the FA core complex (B), where FANCL is the E3 pEAK8-FLAG-FANCE or no DNA (ND). Cells were harvested Mapping these sites of interaction will generate important ligase, UBE2T is the E2 ubiquitin-conjugating enzyme, and and lysed in 2% SDS lysis buffer, and analyzed via Western mechanistic insight into the regulation of this critical tumor FANCE is the substrate (FANCD2) recognition protein. blotting using anti-FLAG and anti-tubulin antibodies. suppressor pathway.

This work was supported by grants from the NIH/NHLBI, the Department of Defense, and the University of Rhode Island Undergraduate Research Initiative