Recombinant ATM protein complements the cellular A-T phenotype

Area of Science:

• Genetics and Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Ataxia-telangiectasia (A-T) is an autosomal recessive disorder with neurodegeneration, immunodeficiency, and cancer predisposition.
• Cellular defects in A-T involve signal transduction pathways crucial for cell cycle checkpoints and response to DNA damage.
• The ATM gene product is a large protein family member essential for genome stability and cell cycle progression.

Purpose of the Study:

• To construct and analyze a full-length recombinant ATM protein for functional studies.
• To develop a tool for investigating ATM protein function in Ataxia-telangiectasia.
• To assess the ability of recombinant ATM to correct the cellular phenotype of A-T cells.

Main Methods:

• Constructed and cloned a full-length ATM open reading frame using a combination of vectors and hosts.
• Expressed recombinant ATM in insect cells via baculovirus vector and in human A-T cells using an episomal vector.
• Utilized an N-terminal FLAG epitope for detection, isolation, and functional analysis of recombinant ATM.

Main Results:

• Recombinant ATM was stably expressed in both insect and human A-T cells.
• Ectopic expression of ATM in A-T cells restored normal ionizing radiation sensitivity and post-irradiation DNA synthesis.
• A specific A-T missense mutation (Glu2904Gly) in recombinant ATM led to protein instability and failed to complement the A-T phenotype.

Conclusions:

• The recombinant, epitope-tagged ATM protein is functional and can correct the cellular defects in Ataxia-telangiectasia.
• The physiological defects in A-T cells are due to the absence of functional ATM protein.
• Ectopic expression of ATM can correct the deficiency characteristic of Ataxia-telangiectasia cells.