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

Abstract

Two major DNA double-strand break repair pathways exist in all eukaryotes, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Although both pathways can function throughout the cell cycle, NHEJ predominates in G0͞G1 (when a replicated sister chromatid is unavailable), whereas HR makes a more substantial contribution in S and G2. How a cell chooses between these two important DNA repair pathways is largely unknown. DNA-dependent protein kinase (DNA-PK) is critical for NHEJ. Here, we describe two conserved splice variants of a catalytic subunit of DNA-PK (DNA-PKcs) that are expressed predominately in nondividing cells. Although both encode stable products, neither reverses the NHEJ defects in DNA-PKcs-deficient cells. In fact, cells expressing one of the DNA-PKcs variants are slightly more radiosensitive than cells completely deficient in DNA-PKcs. We investigated whether cells expressing the DNA-PKcs variants had any other DNA repair deficits and found that these cells are considerably more sensitive to both etoposide and mitomycin C than cells that express no DNA-PKcs at all. Because repair of DNA damage induced by these two agents requires intact HR, we tested whether the NHEJ-defective variants of DNAPKcs inhibit double-strand break-induced HR in an integrated substrate. In cells expressing the NHEJ-defective variants, HR was markedly reduced. Because the splice variants are expressed highly only in nondividing cells, quiescent cells would be afforded a mechanism to inhibit repair by means of HR when sister chromatids are not available as templates for accurate repair with low risk of genome rearrangement, thereby enhancing genome stability.DNA repair ͉ DNA-dependent protein kinase ͉ nonhomologous DNA end joining pathway

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

“…In cells, the effects of DNA-PKcs inhibitors on DNA-PKcs protein levels and NHEJ efficiency, as well as the phosphorylation of DNAPKcs by ATM, likely mask the stimulatory effects of DNA-PKcs catalytic activity on resection and yield conflicting observations (28,29,31,56). Our results in vitro are consistent with previous observations that loss of DNA-PKcs catalytic activity (through truncation of the catalytic domain, chemical inhibition, or mutation of autophosphorylation sites) actually inhibits HR (29,30).…”

“…DNA-PKcs has been shown to possess dual functions in the regulation of both NHEJ and HR (24,(27)(28)(29)(30), but the mechanism by which HR is regulated by DNA-PKcs is not well understood. Our previous study using the cellular resection assay clearly showed that loss of DNA-PKcs leads to increased DSB resection, 3 consistent with the observation that complementation of DNA-PKcs in V3 DNA-PKcs null CHO cells decreases Rad51 foci formation upon DNA damage (24).…”

PRKDC mutations in a SCID patient with profound neurological abnormalities

“…Collectively, these findings provide strong evidence that loss of exon 16 dramatically affects function and likely represents a null mutational change. This finding is consistent with our previous DNA-PKcs mutational studies showing that small N-terminal deletions completely ablate function and that other exon-skipped DNA-PKcs variants are similarly nonfunctional (40,41).…”

“…Our previous studies have shown that PRKDC does not tolerate deletions (40,41). Furthermore, the deleted region is highly conserved in vertebrate PRKDC and, from the available structural studies, is predicted to reside within the N-terminal "pincer" domain.…”

GANP Regulates the Choice of DNA Repair Pathway by DNA-PKcs Interaction in AID-Dependent IgV Region Diversification

“…Many factors, including DNA-PKcs, can regulate the mechanistic overlap or competition between NHEJ and HR (16,(53)(54)(55)68). DNA-PKcs suppresses both spontaneous and DSB-induced HR at the initial step of DNA repair (56).…”