Defining the Sites of Interaction of the FANCD2, FANCE, and FANCL proteins

Author(s)

Joseph McClanaghan, University of Rhode IslandFollow

Major

Microbiology

Advisor

Howlett, Niall, G

Advisor Department

Cell and Molecular Biology

Date

5-2014

Keywords

Fanconi anemia, DNA repair, ubiquitination, chromosome stability, cancer

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

Abstract

Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure and increased cancer susceptibility. FA is caused by mutations in any one of 16 genes. These genes encode for proteins that function in the FA-BRCA pathway to repair damaged DNA. Because of its important r­­­ole in DNA repair, this pathway is considered a major cellular tumor suppressor pathway, i.e. is critical for the prevention of cancer. Underscoring this fact, several of the FA genes - including BRCA2, BRIP1, PALB2, and RAD51C - are bona fide breast and ovarian cancer susceptibility genes.

My project involves studying protein-protein interactions at the key step that localizes two proteins in the pathway (FANCD2 and FANCI) to chromatin. This step, the monoubiquitination of FANCD2 and FANCI is the essential step for repair – one of the simplest methods to diagnose FA is to test for FANCD2 monoubiquitination. Monoubiquitination refers to the addition of a single ubiquitin moiety to a larger protein. The addition of ubiquitin(s) acts as a molecular signal that can elucidate a diverse array of molecular functions, such as protein degradation and nuclear foci formation.

Monoubiquitination is catalyzed by three enzymes: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase. The E1 binds to ubiquitin and transfers it to the E2 ubiquitin-conjugating enzyme. The E3 ubiquitin ligase then transfers the ubiquitin to the substrate for ubiquitination. In the FA-BRCA pathway, UBE2T and FANCL are the E2 and E3, respectively. FANCL falls into the RING E3 ligase family. RING E3 ligases do not physically bind to ubiquitin but rather catalyze the transfer of ubiquitin from the E2 to the substrate.

The major objective of my project is to map the sites of interaction between FANCD2, FANCL, and FANCE. To begin, the necessary DNA reagents need to be developed and tested. We have modified our proteins by placing a small protein tag that allows us to isolate and analyze them. We have also generated four overlapping FANCD2 protein fragments allowing us to determine which region of FANCD2 interacts with the other proteins.

To determine if these proteins interact, and to determine which region of FANCD2 is important for interaction, V5-FANCD2 and HA-FANCL or FLAG-FANCE will be co-transfected into COS-7 African Green Monkey cells, and co-immunoprecipitation experiments will be performed. Whole-cell lysates will be incubated with anti-V5 agarose, a polymer gel matrix used in affinity chromatography of proteins. The agarose recognizes V5 tagged FANCD2 and any proteins in complex with FANCD2. The V5 agarose is then washed to remove proteins that bind non-specifically. The FANCD2 immune complex proteins are then separated by gel electrophoresis and analyzed using Western blotting.

These experiments will allow us to determine if FANCD2, FANCL, and FANCE directly interact, and which region(s) of FANCD2 mediates these interactions. It is hoped that these results will uncover novel mechanistic insights into the biochemistry of an important tumor suppressor pathway.