Insight into the Regulation of the Fanconi Anemia D2 protein, a Major DNA Repair Protein
Phosphorylation; DNA repair; Cancer Biology
Fanconi anemia (FA) is a rare genetic disease characterized by birth defects, bone marrow failure and increased cancer risk. FA is caused by mutation of 22 genes. The proteins coded by these genes function in the FA pathway, which repairs DNA damage, and maintains chromosome stability. Following exposure to DNA damaging agents, the pathway is activated, which leads to the transfer of a small signaling protein, ubiquitin, onto FANCD2. How this important step is regulated remains poorly understood.
Using in silico molecular modeling, we recently identified four putative phosphorylation sites proximal to the site of FANCD2 monoubiquitination, lysine 561 (K561). Phosphorylation is another form of protein modification, and involves the replacement of a hydroxyl group in the side chain of an amino acid (serine (S), threonine (T), or tyrosine (Y)) with a phosphate group. Phosphates are added to proteins by enzymes called kinases, and removed by phosphatases. The newly identified phosphorylation sites are candidate cyclin-dependent kinase (CDK) sites, kinases that play a major role in regulating cell cycle progression. We hypothesize that FANCD2 is phosphorylated by CDKs, thereby regulating its activity during the cell cycle.
The goal of my honors project was to determine if FANCD2 is phosphorylated by CDKs. We planned to test this hypothesis by purifying a fragment of FANCD2 containing the four-phosphorylation sites and performing an in vitro CDK kinase assay. First, I used molecular visualization software to design a fragment of FANCD2 containing the four-phosphorylation sites, without disrupting protein secondary structure. I then designed PCR primers to amplify this region and to clone this fragment into the pET-28a plasmid using restriction enzyme cloning. Two plasmids were generated: pET-28a-FANCD2-WT, containing the wild-type FANCD2 sequence, and pET-28a-FANCD2-TA, containing the FANCD2 sequence mutated at the putative phosphorylation sites.
Immobilized metal affinity chromatography will be used to purify the FANCD2-WT and FANCD2-TA proteins. I will then incubate these purified proteins with several different cyclin-CDK pairs to determine if FANCD2 is phosphorylated by CDK and if mutation of the putative phosphorylation sites blocks phosphorylation. We anticipate that these experiments will greatly improve our understanding of the regulation of the FANCD2 protein.