53BP1 dependent non-homologous end-joining pathway as a backup for G1 phase DNA double-strand-break repair

Abstract

To investigate a role of 53BP1 in repair of DNA double-strand breaks(DSBs) in vertebrate cells, we established, using hyperrecombinogenic chicken B cell line DT40, a 53BP1 knockout cell line and double knockout cell lines deficient in 53BP1 and either Ku, DNA ligase IV (Lig IV), ATM or Artemis. 53BP1-/- cells showed intact intra-S and G2/M phase checkpoint for DNA DSBs. We carried out an epistasis analysis, using colony formation assay in G1 synchronized cells, and revealed that there are at least three sub-pathways in non-homologous end-joining (NHEJ), 1) the core NHEJ which is dependent on Ku/DNA-PK, 2) Artemis dependent pathway which includes ATM, and 3) 53BP1-dependent pathway. These three pathways lead to Lig IV in the final ligation step. ATM deficiency showed only a marginal effect on the colony formation, and Artemis was modulated by DNA-PK. In contrast to core NHEJ and Artemis dependent pathway, 53BP1 dependent pathway was resistant to PI-3 kinase inhibitor Wortmannin, and showed high contribution to DSB repair only in G1, but not in early S phase.