Backup pathways of NHEJ are suppressed by DNA-PK

In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA-PK) and is therefore termed here D-NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B-NHEJ). Here, we investigate the role of DNA-PK in the functional coordination of D-NHEJ and B-NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA-PK, the degree of inhibition depends on the ratio between DNA ends and DNA-PK, suggesting that binding of inactive DNA-PK to DNA ends not only blocks processing by D-NHEJ, but also prevents the function of B-NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA-PK, is attributed to the function of B-NHEJ operating on DNA ends free of DNA-PK. Thus, DNA-PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D-NHEJ.