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Updated: Aug 2, 2026
CD Spectroscopy to Study DNA-Protein Interactions
Published on: February 10, 2022
Purification and DNA binding properties of the ataxia-telangiectasia gene product ATM
Abstract:
The human neurodegenerative and cancer predisposition condition ataxia-telangiectasia is characterized at the cellular level by radiosensitivity, chromosomal instability, and impaired induction of ionizing radiation-induced cell cycle checkpoint controls. Recent work has revealed that the gene defective in ataxia-telangiectasia, termed ATM, encodes an approximately 350-kDa polypeptide, ATM, that is a member of the phosphatidylinositol 3-kinase family. We show that ATM binds DNA and exploit this to purify ATM to near homogeneity. Atomic force microscopy reveals that ATM exists in two populations, with sizes consistent with monomeric and tetrameric states. Atomic force microscopy analyses also show that ATM binds preferentially to DNA ends. This property is similar to that displayed by the DNA-dependent protein kinase catalytic subunit, a phosphatidylinositol 3-kinase family member that functions in DNA damage detection in conjunction with the DNA end-binding protein Ku. Furthermore, purified ATM contains a kinase activity that phosphorylates serine-15 of p53 in a DNA-stimulated manner. These results provide a biochemical assay system for ATM, support genetic data indicating distinct roles for DNA-dependent protein kinase and ATM, and suggest how ATM may signal the presence of DNA damage to p53 and other downstream effectors.
Insights
Ataxia-telangiectasia mutated (ATM) protein binds DNA, particularly at DNA ends, and possesses kinase activity. This discovery provides a biochemical assay for ATM and suggests its role in DNA damage signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Ataxia-telangiectasia (A-T) is a neurodegenerative and cancer predisposition disorder.
- Cellular hallmarks of A-T include radiosensitivity and impaired cell cycle checkpoint control after ionizing radiation.
- The gene responsible for A-T, ATM, encodes a phosphatidylinositol 3-kinase family member.
Purpose of the Study:
- To biochemically characterize the ATM protein.
- To investigate ATM's interaction with DNA.
- To elucidate ATM's role in DNA damage response pathways.
Main Methods:
- Purification of ATM protein using DNA-binding properties.
- Atomic force microscopy (AFM) to analyze ATM structure and DNA binding.
- In vitro kinase assays to assess ATM's enzymatic activity.
Main Results:
- Purified ATM protein binds DNA preferentially to DNA ends.
- AFM revealed ATM exists as monomers and tetramers.
- ATM exhibits DNA-stimulated kinase activity, phosphorylating p53 at serine-15.
Conclusions:
- ATM's DNA-binding and kinase activities provide a basis for biochemical assays.
- ATM's function at DNA ends suggests a role in DNA damage detection.
- ATM likely signals DNA damage to downstream effectors like p53.
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