Autophosphorylation of the Catalytic Subunit of the DNA-Dependent Protein Kinase Is Required for Efficient End Processing during DNA Double-Strand Break Repair

Abstract

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.Although DNA is the genetic blueprint for all living organisms, it is extremely sensitive to various forms of damage, including oxidation, hydrolysis, and methylation. Thus, efficient DNA repair systems are essential for the maintenance of chromosomal integrity. DNA double-strand breaks (DSBs) are perhaps the most lethal form of DNA damage. In eukaryotes, primarily two pathways repair DSBs: homologous recombination and nonhomologous DNA end joining (NHEJ). In higher eukaryotes, NHEJ is thought to be the major pathway that repairs these breaks (6,29,37,38). Because NHEJ also functions in developing lymphocytes to repair the DSBs introduced during antigen receptor gene rearrangement, defects in this pathway result in a block in lymphocyte development and the disease known as severe combined immunodeficiency (SCID) (reviewed in references 14 and 29).In the past decade, an intensive research effort has focused on NHEJ, resulting in a reasonable understanding of how DSBs are resolved. There are six known factors which unequivocally function in the NHEJ pathway. Three of these comprise the DNA-dependent protein kinase (DNA-PK) (reviewed in references 19, 20, and 25): the two subunits of the DNA end binding heterodimer Ku and the catalytic subunit of DNA-PK (DNA-PKcs) (18). Two other factors, XRCC4 and DNA ligase IV, form a stable DNA ligase complex (15,28,34). The sixth factor, Artemis, was described in 2001 (35); recent data indicate that it may play an important role in DNA end processing during NHEJ (31).DNA-PK plays a central role in NHEJ because it initially recognizes and binds to damaged DNA and then targets other...

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.…”

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).…”

Non-homologous end-joining, a sticky affair

“…DNA end bound Ku70/80 then attracts the catalytic subunit of the DNA-dependent protein kinase (DNA-PK CS ) and activates its protein kinase activity (Smith and Jackson, 1999). Although many different targets have been identified, the major function for this phosphorylation activity appears to be regulation of the NHEJ reaction by DNA-PK CS autophosphorylation (Chan et al, 2002;Ding et al, 2003;Weterings et al, 2003). This reaction takes place after juxtaposition of DNA ends and is required for proper regulation of DNA end accessibility for other NHEJ proteins (Meek et al, 2007).…”

“…The assembled DNA-PK complex then acquires the ability to phosphorylate a number of target proteins, but the functional relevance of most targets is not clear. However, DNA-PK CS autophosphorylation is clearly important for the NHEJ reaction (Chan et al, 2002;Ding et al, 2003;Weterings et al, 2003). It is accomplished in trans, after formation of a synaptic complex that brings together the two sides of the DSB (Meek et al, 2007).…”

“…Several clusters of autophosphorylation sites have been mapped. A cluster of seven sites between residues 2609 and 2647 has a major influence on remodeling the DNA-PK complex to make the DNA ends accessible for ligation (Ding et al, 2003;Block et al, 2004;Reddy et al, 2004). Conversely, phosphorylation in the region between residues 2023-2056 appears to be involved in reducing access of nuclease activities to the DNA ends Cui et al, 2005), while phosphorylation of residue 3950 also influences the joining efficiency .…”