Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends

Abstract

Non-homologous end joining (NHEJ) is one of the primary pathways for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in mammalian cells. Proteins required for NHEJ include the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), Ku, XRCC4 and DNA ligase IV. Current models predict that DNA-PKcs, Ku, XRCC4 and DNA ligase IV assemble at DSBs and that the protein kinase activity of DNA-PKcs is essential for NHEJ-mediated repair of DSBs in vivo. We previously identified a cluster of autophosphorylation sites between amino acids 2609 and 2647 of DNA-PKcs. Cells expressing DNA-PKcs in which these autophosphorylation sites have been mutated to alanine are highly radiosensitive and defective in their ability to repair DSBs in the context of extrachromosomal assays. Here, we show that cells expressing DNA-PKcs with mutated autophosphorylation sites are also defective in the repair of IR-induced DSBs in the context of chromatin. Purified DNA-PKcs proteins containing serine/threonine to alanine or aspartate mutations at this cluster of autophosphorylation sites were indistinguishable from wild-type (wt) protein with respect to protein kinase activity. However, mutant DNA-PKcs proteins were defective relative to wt DNA-PKcs with respect to their ability to support T4 DNA ligase-mediated intermolecular ligation of DNA ends. We propose that autophosphorylation of DNA-PKcs at this cluster of sites is important for remodeling of DNA-PK complexes at DNA ends prior to DNA end joining.

Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

“…Thus, blocking DNA-PK's kinase activity inhibits both DSB-induced and spontaneous HR (26); inhibits telomere maintenance (38); and inhibits the repair of damage from topoisomerase II poisons (39). Emerging data suggest that DNA-PK that cannot undergo autophosphorylation (either by abrogating its kinase activity or by abrogating its autophosphorylation sites) may remain associated with DNA ends, thus preventing access to other repair factors (16,18,19,40,41). This hypothesis is a plausible explanation for how inactive DNA-PK inhibits other repair pathways.…”

The life and death of DNA-PK

“…DNA-PK cs is autophosphorylated after cellular IR exposures (Chan et al, 2002;Douglas et al, 2002;Ding et al, 2003;Block et al, 2004b;Lou et al, 2004), which modifies its binding with Ku and has been implicated in the phosphorylation of a wide range of DNA damage/checkpoint proteins. This auto-phosphorylation event is critical for correct NHEJ activity within the cell (Chan et al, 2002;Block et al, 2004b). Recent work by Lou et al (2004) has shown that the mediator of DNA damage checkpoint protein 1 (MDC1), which is generally associated with the regulation of both intra S-phase and G2/M phase checkpoints, directly binds to DNA-PK via repeat regions and augments these early auto-phosphorylation events.…”

Regulation of DNA-dependent protein kinase by protein kinase CK2 in human glioblastoma cells

“…In this respect, it has been reported that DNA-PKcs can undergo autophosphorylation (Chan et al, 2002;Douglas et al, 2002Douglas et al, , 2007Chen et al, 2005). To date, it is not clear which is the exact role of this event, although some studies have reported that autophosphorylation of purified DNAPKcs results in disruption of the DNA-PK complex in vitro and loss of kinase activity in vivo (Chan and Lees-Miller, 1996;Merkle et al, 2002;Ding et al, 2003;Block et al, 2004;Douglas et al, 2007). This leaves open the possibility that dissociation of the DNA-PK complex from DNA might be necessary for facilitating subsequent repair steps by allowing the assembly of damage-responsive proteins to the site of DNA damage or activating them.…”