Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

Division of Molecular Radiation Biology, Department of Radiation Oncology, The University of Texas Southwestern Medical Center, 2201 Inwood Rd, Dallas, TX 75390, USA.
DNA repair (Impact Factor: 3.11). 03/2012; 11(3):310-6. DOI: 10.1016/j.dnarep.2011.12.007
Source: PubMed


DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.

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    • "The participation of PARP3 in the recruitment of Ku80 and its poly(ADP-ribosyl)ation reported here define PARP3 as a key player in the assembly of Ku complexes during C-NHEJ. As proposed above, this would also help to prevent extensive resection during HR or A-EJ as it was suggested for Ku80 (53,80). However, the exact molecular mechanism involved remains to be clarified. "
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    ABSTRACT: The repair of toxic double-strand breaks (DSB) is critical for the maintenance of genome integrity. The major mechanisms that cope with DSB are: homologous recombination (HR) and classical or alternative nonhomologous end joining (C-NHEJ versus A-EJ). Because these pathways compete for the repair of DSB, the choice of the appropriate repair pathway is pivotal. Among the mechanisms that influence this choice, deoxyribonucleic acid (DNA) end resection plays a critical role by driving cells to HR, while accurate C-NHEJ is suppressed. Furthermore, end resection promotes error-prone A-EJ. Increasing evidence define Poly(ADP-ribose) polymerase 3 (PARP3, also known as ARTD3) as an important player in cellular response to DSB. In this work, we reveal a specific feature of PARP3 that together with Ku80 limits DNA end resection and thereby helps in making the choice between HR and NHEJ pathways. PARP3 interacts with and PARylates Ku70/Ku80. The depletion of PARP3 impairs the recruitment of YFP-Ku80 to laser-induced DNA damage sites and induces an imbalance between BRCA1 and 53BP1. Both events result in compromised accurate C-NHEJ and a concomitant increase in DNA end resection. Nevertheless, HR is significantly reduced upon PARP3 silencing while the enhanced end resection causes mutagenic deletions during A-EJ. As a result, the absence of PARP3 confers hypersensitivity to anti-tumoral drugs generating DSB.
    Full-text · Article · Mar 2014 · Nucleic Acids Research
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    • "However, removal of all putative Cdk1 phosphorylation sites on Ku70 and 3 out of 4 sites on Ku80 failed to elicit any DSB repair phenotype in S. cerevisiae, suggesting that the negative regulation of Ku by Cdk1 is most likely indirect (Zhang et al., 2009). Ku binding to DNA ends also attenuates resection and HR in mammalian cells (Shao et al., 2012; Tomimatsu et al., 2012). Furthermore, Ku70 was reported as a binding partner and substrate of CDK2-cyclin A, and Ku70-T455 was identified as a CDK target site by mass spectrometry (Müller-Tidow et al., 2004; Chi et al., 2008; Olsen et al., 2010); but whether or not Ku phosphorylation by CDKs has an impact on DNA-end resection has yet to be determined. "
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    ABSTRACT: DNA double-strand breaks (DSBs) are repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). The choice between HR and NHEJ is highly regulated during the cell cycle. DNA-end resection, an evolutionarily conserved process that generates long stretches of single-stranded DNA, plays a critical role in pathway choice, as it commits cells to HR, while, at the same time, suppressing NHEJ. As erroneous DSB repair is a major source of genomic instability-driven tumorigenesis, DNA-end resection factors, and in particular their regulation by post-translational modifications, have become the subject of extensive research over the past few years. Recent work has implicated phosphorylation at S/T-P motifs by cyclin-dependent kinases (CDKs) as a major regulatory mechanism of DSB repair. Intriguingly, CDK activity was found to be critically important for the coordinated and timely execution of DNA-end resection, and key players in this process were subsequently identified as CDK substrates. In this mini review, we provide an overview of the current understanding of how the DNA-end resection machinery in yeast and human cells is controlled by CDK-mediated phosphorylation.
    Full-text · Article · Jun 2013 · Frontiers in Genetics
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    • "Ku plays multiple roles during NHEJ. Initially, Ku not only binds and detects DSB but also protects the ends from nucleolytic degradation and tilts the choice of DSB repair towards NHEJ and away from HR [7] [36]. Perhaps the most extensive role for Ku is to recruit NHEJ factors to DSB sites. "

    Full-text · Chapter · May 2013
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