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J Biomol Tech. 2007 February; 18(1): 53.
PMCID: PMC2292033

P153-T DNA Damage Induces a Novel Interaction between Fanca and Huntingtin

Abstract

The objective of this study was to elucidate the role that Fanconi anemia complementation group A protein (FANCA) plays in cells upon DNA damage through identification of DNA damage dependent protein interactions with FANCA using the Tandem Affinity Purification (TAP) methodology. Fanconi anemia (FA) is a rare autosomal recessive disease that is characterized by aplastic anemia, congenital abnormalities, birth defects and increased susceptibility to cancer. FA patients are assigned to one of twelve complementation groups, with about 65% of patients falling into complementation group A. FA patients share the common phenotypic feature of FA of bone marrow failure, and cells from FA patient’s exhibit sensitivity to DNA crosslinking agents such as Mitomycin C (MMC) and Diepoxybutane. TAP was used in order to purify the protein complexes twice, and to maintain protein interactions based on post translational modifications. The TAP FANCA construct contains two tags, a streptavidin binding peptide (SBP) and a calmodulin binding peptide (CBP) which allow for the two-step purification. Human embryonic kidney cells (HEK 293) were transiently transfected with either a negative control or the TAP FANCA construct. Half of the FANCA transfected HEK 293 plates were treated with MMC twenty-four hours post transfection to induce DNA damage. The transfected cells were collected forty-eight hours post transfection, and the FANCA interacting proteins were TAP purified and identified using Mass Spectrometry. MMC treatment induced a novel interaction between FANCA and Huntingtin (HTT) that was not found in the untreated FANCA or negative control samples. The DNA damaged induced FANCA/HTT interaction was confirmed through western blot. The role of the FANCA/HTT interaction in DNA damage is not known. More studies are underway to determine if this interaction is important for DNA damage recognition or repair.


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