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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

ABSTRACT 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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    • "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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