CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

Radiation Biology and DNA Repair, Darmstadt University of Technology, 64287 Darmstadt, Germany.
Nucleic Acids Research (Impact Factor: 9.11). 11/2010; 39(6):2144-52. DOI: 10.1093/nar/gkq1175
Source: PubMed


Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through
it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction
and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5′-end of the DNA. Such DSBs are repaired
by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior
to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1
in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends.
Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN
complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the
Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes
that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its
resection function.

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    • "In addition, the MRN complex localizes to the telomeres and regulates telomeric length either by recruitment of the telomerase RNA subunit or as a sensor of damaged telomeres promoting ATM activation and alternative lengthening of telomeres [132] [133] [134]. The MRN-mediated end resection and utilization of both HR and NHEJ pathways is promoted by CtIP protein [135] [136] [137]. CtIP has recently been shown to possess nuclease activity required for processing DNA adducts or secondary structures at the sites of breaks [138] [139], making it also possible target for chemical intervention. "
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    ABSTRACT: Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.
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    • "APE1 has been implicated in repair of bleomycin-induced DSBs (8), whereas cytogenetic and survival assays suggest a role for TDP1 in resolution of 3′-PG termini of calicheamicin-induced DSBs (9). In the case of topoisomerase II-mediated DSBs, survival and focus-formation assays suggest that TDP2 and CtIP are each involved in separate pathways for removal of the 5′-tyrosyl-linked TOP2 fragments at TOP2-mediated DSBs (10,11). "
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    • "All other siRNAs were purchased from Eurofins MWG Operon. siRNA sequences are as follows: siControl: AAUUCUCCGAACGUGUCACGUdTdT (26); siCtIP: GCUAAAACAGGAACGAAUCdTdT (4); siMre11: ACAGGAGAAGAGAUCAACUdTdT (26); siSOSS-A:CGUGAUGGCAUGAAUAUUGdTdT (27); siExo1: UAGUGUUUCAGGAUCAACAUCAUCUdTdT (28); siDNA-PKcs: CUUUAUGGUGGCCAUGGAGdTdT (29); siBRCA1: GGAACCUGUCTCCACAAAGdTdT (30); si53BP1: GGACUCCAGUGUUGUCAUUdTdT (31). The efficiency of gene knockdown was examined by western blotting and DSB resection was measured 48 h after transfection. "
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    ABSTRACT: 5′ strand resection at DNA double strand breaks (DSBs) is critical for homologous recombination (HR) and genomic stability. Here we develop a novel method to quantitatively measure single-stranded DNA intermediates in human cells and find that the 5′ strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle–dependent manner. Depletion of CtIP, Mre11, Exo1 or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of Brca1 does not, suggesting that the role of Brca1 in HR is primarily to promote Rad51 filament formation, not to regulate end resection.
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