ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• H2AX phosphorylation is a critical early event in DNA damage response.
• ATM is the established kinase for H2AX phosphorylation following DNA double-strand breaks (DSBs).
• The role of DNA-dependent protein kinase (DNA-PK) in H2AX phosphorylation remains unclear.

Purpose of the Study:

• To investigate the role of DNA-PK in H2AX phosphorylation induced by ionizing radiation (IR).
• To elucidate the interplay between ATM and DNA-PK in the DNA damage response pathway.
• To determine if DNA-PK contributes to the recruitment of DNA repair proteins to DSBs.

Main Methods:

• Utilized human fibroblasts, mouse embryo fibroblasts (lacking DNA-PK or ATM), and chicken cells.
• Exposed cells to ionizing radiation (IR) to induce DNA double-strand breaks (DSBs).
• Employed LY294002, a specific DNA-PK inhibitor, to assess its impact on H2AX phosphorylation.

Main Results:

• H2AX phosphorylation occurred similarly in cells lacking DNA-PK or ATM, but was abolished in ATM-deficient cells treated with a DNA-PK inhibitor.
• Complete ablation of IR-induced H2AX phosphorylation required inactivation of both DNA-PK and ATM in chicken cells.
• H2AX phosphorylation by DSBs in non-replicating cells was independent of ATR.
• DNA-PK, while unable to phosphorylate other checkpoint proteins, contributes to MDC1 and 53BP1 recruitment to DSBs via H2AX phosphorylation.

Conclusions:

• ATM and DNA-PK act in a redundant and overlapping manner to phosphorylate H2AX under normal growth conditions following IR.
• DNA-PK plays a significant role in H2AX phosphorylation and subsequent recruitment of DNA damage response proteins to DSBs.
• This redundant pathway involving ATM and DNA-PK is crucial for efficient DNA double-strand break repair initiation.