Backup pathways of NHEJ are suppressed by DNA‐PK

Abstract

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.

Linking double-stranded DNA breaks to the recombination activating gene complex directs repair to the nonhomologous end-joining pathway

“…Although we have not formally shown that the I-Sce1 breaks in this experiment are chromatinized, these data suggest that targeting breaks to a chromatin compartment does not ensure repair by NHEJ. In summary, we suggest, consistent with previous reports (13,14), that alternative end-joining pathways exist, but to date are not well characterized (19)(20)(21)(22)(23) and can efficiently rejoin I-Sce1 breaks in the absence of NHEJ, albeit with reduced fidelity. A previous report, studying rejoining of a chromosomal I-Sce1 substrate in Ku-deficient cells, made essentially the same conclusion (24).…”

“…Several reports have documented a variety of NHEJ subpathways, some which are independent of the DNA-PK and XRCC4/ ligase IV complexes (18)(19)(20)(21)(22). One of these pathways may mediate the efficient plasmid rejoining observed in NHEJdefective cells.…”

Two distinct cytokinesis pathways drive trypanosome cell division initiation from opposite cell ends

“…In the absence of the canonical NHEJ (c-NHEJ), cells can repair a large portion of DSBs using an alternative pathway that employs a distinct set of factors (27). This alternative NHEJ, termed alt-NHEJ or b-NHEJ (for backup NHEJ), operates with an order of magnitude slower kinetics, frequently joins incorrect ends, and is suppressed by the c-NHEJ (28,29). Additional examples of the existence of a backup pathway were also found in other cellular processes, such as cell death (30), endoplasmic reticulum-associated degradation (31), and mRNA decay (32).…”

DNA double-strand break repair and development

“…While NHEJ requires DNA-PK and Lig4, studies suggest that there may be NHEJ repair that utilizes other components, and in some cases can occur without DNA-PK (DiBiase et al, 2000;Perrault et al, 2004). For instance, DNA ligase III (Lig3) has been implicated as a alternate ligase that may function in NHEJ (Wang et al, 2005a;Windhofer et al, 2007).…”