ATR autophosphorylation as a molecular switch for checkpoint activation

Area of Science:

• Molecular Biology
• Cellular Biology
• Genetics

Background:

• The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is essential for maintaining genomic stability.
• ATR activation following DNA damage is a complex process involving the ATR-ATRIP complex and RPA-coated single-stranded DNA (ssDNA).

Purpose of the Study:

• To elucidate the precise mechanism of ATR kinase activation upon DNA damage.
• To identify the key molecular events and interactions governing ATR activation.

Main Methods:

• Investigated ATR phosphorylation states using biochemical assays.
• Utilized site-directed mutagenesis to probe the role of specific phosphorylation sites, particularly Thr 1989.
• Examined protein-protein interactions between ATR, ATRIP, RPA, and TopBP1 using various biochemical and biophysical techniques.

Main Results:

• DNA damage induces hyperphosphorylation of ATR, with autophosphorylation at Thr 1989 being critical for activation.
• ATR activation at Thr 1989 requires RPA, ATRIP, and ATR kinase activity, but not TopBP1 directly.
• Recruitment of ATR-ATRIP to RPA-ssDNA promotes trans-phosphorylation at Thr 1989.
• Phosphorylated Thr 1989 serves as a recognition site for TopBP1, facilitating ATR stimulation and substrate engagement.

Conclusions:

• ATR autophosphorylation at Thr 1989 acts as a molecular switch for initiating a robust DNA damage checkpoint response.
• The findings reveal a novel mechanism for ATR activation, highlighting the sequential roles of RPA, ATRIP, ATR, and TopBP1.