Abstract
Nonhomologous end joining (NHEJ) is one of the major pathways that mediate the repair of DNA double-strand breaks (DSBs) in mammalian cells. The DNA-dependent protein kinase (DNA-PK) complex plays important roles in this pathway, since mammalian cell lines that lack components of the complex are radiation sensitive and are defective in rejoining DSBs. DNA-PKcs is a serine/threonine protein kinase that is activated by DNA ends in vitro and that consists of the DNA-binding and regulatory subunit, Ku, and the catalytic subunit, DNA-PKcs. DNA-PK kinase activity is required for the repair of DNA DSBs in vivo; however, the molecular basis of this requirement is unknown. DNA-PKcs (DNA-PK catalytic subunit) is phosphorylated at various sites in vivo in response to ionizing radiation and the phosphophrylated DNA-PKcs is localized at the DNA double-strand breaks. Furthermore, mutations prevent DNA-PKcs phosphorylation lead to radiation sensitivity and impaired DSB rejoining, suggesting that phosphorylation of DNA-PKcs is required for the repair of DSBs by NHEJ pathway. Interestingly, IR-induced DNA-PKcs phosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs result in robust DNA-PKcs phosphorylation and localization to DNA damage sites. These results indicate for the first time that, while IR-induced DNA-PKcs phosphorylation is attenuated in S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis. The possible role of DNA-PK and NHEJ in repair of replication-associated DNA double-strand breaks in S phase will be discussed.
