The Mismatch-Binding Factor MutSβ Can Mediate ATR Activation in Response to DNA Double-Strand Breaks

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• The Ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is crucial for DNA damage response.
• ATR activation is triggered by RPA-coated single-stranded DNA (ssDNA) at stalled replication forks or DNA double-strand breaks (DSBs).

Purpose of the Study:

• To identify novel regulators of ATR activation by ssDNA.
• To elucidate the mechanism by which MutSβ interacts with ATR signaling.

Main Methods:

• Depletion of MSH2 and MSH3 in cells and assessment of ATRIP foci formation and ATR substrate phosphorylation.
• Biochemical assays using purified MutSβ to assess binding to RPA-ssDNA complexes and hairpin structures.
• Site-directed mutagenesis of MSH3 to investigate the role of its mismatch-binding domain.

Main Results:

• MSH2 and MSH3 form a complex with ATR and ATRIP, and their depletion impairs ATR activation.
• Purified MutSβ binds to DNA hairpin loops within RPA-ssDNA complexes.
• Mutations in the MSH3 mismatch-binding domain abolish MutSβ binding to hairpin loops and ATR activation.

Conclusions:

• MutSβ (MSH2-MSH3) acts as a key mediator in ATR activation by ssDNA.
• DNA hairpin structures within ssDNA are recognized by MutSβ and promote ATRIP recruitment.
• This pathway highlights a novel mechanism for triggering DNA damage response signaling.