Abstract
The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.
Highlights
The processing of DNA double strand breaks is critical for homologous recombination
Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1)
To accurately measure single strand DNA (ssDNA) generated by resection adjacent to specific AsiSI-induced double strand breaks (DSBs), we focused on two AsiSI sites on chromosome 1 (“DSB1,” chromosome 1, 89231183; “DSB2,” chromosome 1, 109838221) [38] and designed three pairs of quantitative PCR (qPCR) primers across BsrGI (DSB1) or BamHI (DSB2) restriction sites located various distances from each AsiSI site (DSB1, 335, 1618, and 3500 bp; DSB2, 364, 1754, and 3564 bp) ( Fig. 1A)
Summary
The processing of DNA double strand breaks is critical for homologous recombination. Results: The nonhomologous end joining factor DNA-dependent protein kinase (DNA-PK) blocks end resection but is regulated by autophosphorylation, the Mre11-Rad5-Nbs (MRN) complex, and the ataxia telangiectasia-mutated (ATM) kinase. The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo). This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and DNA repair pathway choice. Eukaryotic cells have developed two major pathways to repair DSBs as follows: non-
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