WASH interacts with Ku to regulate DNA double-stranded break repair

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) is known to regulate gene transcription and nuclear organization.
• Its role in DNA damage response and repair pathways has not been previously elucidated.

Purpose of the Study:

• To investigate the function of WASH in DNA double-stranded break (DSB) repair.
• To elucidate the molecular mechanisms by which WASH participates in the non-homologous end-joining (NHEJ) pathway.

Main Methods:

• Co-immunoprecipitation assays to identify WASH interacting partners.
• Western blotting to assess protein phosphorylation and chromatin relaxation.
• Depletion of WASH using siRNA and assessment of cell sensitivity to etoposide.
• Immunofluorescence microscopy to track recruitment of repair factors to DSBs.

Main Results:

• WASH interacts with core non-homologous end-joining (NHEJ) factors, including Ku70/Ku80 and DNA-PKcs.
• WASH depletion increases cell sensitivity to etoposide-induced DSBs and reduces NHEJ efficiency.
• Loss of WASH inhibits phosphorylation of DNA-PKcs, H2AX, and KAP1, and impairs chromatin relaxation and recruitment of downstream NHEJ factors.
• WASH's function in DSB repair is dependent on its C-terminal VCA domain and Arp2/3 activation.

Conclusions:

• WASH is a novel regulator of DNA DSB repair via the NHEJ pathway.
• WASH facilitates DSB repair by promoting the recruitment and activation of key NHEJ factors.
• These findings reveal a new mechanistic insight into the role of WASH in maintaining genome stability.