Role of mammalian Mre11 in classical and alternative nonhomologous end joining
ABSTRACT The mammalian Mre11-Rad50-Nbs1 (MRN) complex coordinates double-strand break signaling with repair by homologous recombination and is associated with the H2A.X chromatin response to double-strand breaks, but its role in nonhomologous end joining (NHEJ) is less clear. Here we show that Mre11 promotes efficient NHEJ in both wild-type and Xrcc4(-/-) mouse embryonic stem cells. Depletion of Mre11 reduces the use of microhomology during NHEJ in Xrcc4(+/+) cells and suppresses end resection in Xrcc4(-/-) cells, revealing specific roles for Mre11 in both classical and alternative NHEJ. The NHEJ function of Mre11 is independent of H2A.X. We propose a model in which both enzymatic and scaffolding functions of Mre11 cooperate to support mammalian NHEJ.
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ABSTRACT: The actin depolymerizing factor (ADF)/cofilin protein family is essential for actin dynamics, cell division, chemotaxis and tumor metastasis. Cofilin-1 (CFL-1) is a primary non-muscle isoform of the ADF/cofilin protein family accelerating the actin filamental turnover in vitro and in vivo. In response to environmental stimulation, CFL-1 enters the nucleus to regulate the actin dynamics. Although the purpose of this cytoplasm-nucleus transition remains unclear, it is speculated that the interaction between CFL-1 and DNA may influence various biological responses, including DNA damage repair. In this review, we will discuss the possible involvement of CFL-1 in DNA damage responses (DDR) induced by ionizing radiation (IR), and the implications for cancer radiotherapy.International Journal of Molecular Sciences 02/2015; 16(2):4095-4120. DOI:10.3390/ijms16024095 · 2.46 Impact Factor
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ABSTRACT: In concurrent chemoradiotherapy, drugs are used to sensitize tumors to ionizing radiation (IR). Although a spectrum of indications for simultaneous treatment with drugs and radiation has been defined, the molecular mechanisms underpinning tumor radiosensitization remain incompletely characterized for several such combinations. Here, we investigate the mechanisms of radiosensitization by the arabinoside nucleoside analog (NA) 9-β-D-arabinofuranosyladenine (araA) placing particular emphasis on the repair of DNA double-strand-breaks (DSBs), and compare the results to those obtained with fludarabine (F-araA) and cytarabine (araC). Post-irradiation treatment with araA strongly sensitizes cells to IR, but leaves unchanged DSB repair by NHEJ in logarithmically growing cells, in sorted G1- or G2-phase populations, as well as in cells in the plateau-phase of growth. Notably, araA strongly inhibits DSB repair by homologous recombination (HRR), as assessed by scoring IR-induced Rad51 foci, and in functional assays using integrated reporter constructs. Cells compromised in HRR by RNAi-mediated transient knockdown of Rad51 show markedly reduced radiosensitization after treatment with araA. Remarkably, mutagenic DSB repair compensates for HRR inhibition in araA-treated cells. Compared to araA, F-araA and araC are only modestly radiosensitizing under the conditions examined. We propose that the radiosensitizing potential of NAs is linked to their ability to inhibit HRR and concomitantly promote the error-prone processing of DSBs. Our observations pave the way to treatment strategies harnessing the selective inhibitory potential of NAs and the development of novel compounds specifically utilizing HRR inhibition as a means of tumor cell radiosensitization. Copyright © 2015, American Association for Cancer Research.Molecular Cancer Therapeutics 04/2015; DOI:10.1158/1535-7163.MCT-14-0682 · 6.11 Impact Factor
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ABSTRACT: Nonhomologous DNA end joining (NHEJ) is one of the major double-strand break (DSB) repair pathways in higher eukaryotes. Recently, it has been shown that alternative NHEJ (A-NHEJ) occurs in the absence of classical NHEJ and is implicated in chromosomal translocations leading to cancer. In the present study, we have developed a novel biochemical assay system utilizing DSBs flanked by varying lengths of microhomology to study microhomology-mediated alternative end joining (MMEJ). We show that MMEJ can operate in normal cells, when microhomology is present, irrespective of occurrence of robust classical NHEJ. Length of the microhomology determines the efficiency of MMEJ, 5 nt being obligatory. Using this biochemical approach, we show that products obtained are due to MMEJ, which is dependent on MRE11, NBS1, LIGASE III, XRCC1, FEN1 and PARP1. Thus, we define the enzymatic machinery and microhomology requirements of alternative NHEJ using a well-defined biochemical system.Cell Death & Disease 03/2015; 6(3):e1697. DOI:10.1038/cddis.2015.58 · 5.18 Impact Factor