The Mre11/Rad50/Nbs1 complex initiates double-strand break repair by homologous recombination (HR). Loss of Mre11 or its nuclease activity in mouse cells is known to cause genome aberrations and cellular senescence, although the molecular basis for this phenotype is not clear. To identify the origin of these defects, we characterized Mre11-deficient (MRE11) and nuclease-deficient Mre11 (MRE11) chicken DT40 and human lymphoblast cell lines. These cells exhibit increased spontaneous chromosomal DSBs and extreme sensitivity to topoisomerase 2 poisons. The defects in Mre11 compromise the repair of etoposide-induced Top2-DNA covalent complexes, and MRE11 and MRE11 cells accumulate high levels of Top2 covalent conjugates even in the absence of exogenous damage. We demonstrate that both the genome instability and mortality of MRE11 and MRE11 cells are significantly reversed by overexpression of Tdp2, an enzyme that eliminates covalent Top2 conjugates; thus, the essential role of Mre11 nuclease activity is likely to remove these lesions.

Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3 0 single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR. IntroductionDNA double-strand breaks (DSBs) are the most dangerous DNA damage, as a single unrepaired DSB can trigger apoptosis. DSBs are generated during physiological replication and induced by ionizingradiation. DSBs are repaired by two major DSB-repair pathways, homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice of DSB-repair pathway depends on the cell-cycle phase and the DNA-damaging agent (Symington & Gautier 2011). HR repairs DSBs in the S to G 2 phases, whereas NHEJ operates in all the cell phases. HR is more prominent than NHEJ in the repair of DSBs occurring during DNA replication (Hochegger et al. 2006;Qing et al. 2011) and is essential for cellular proliferation. Indeed, loss of critical HR factors, including CtIP, Mre11 and Rad51, causes mortality due to severe genome instability (Yamazoe et al. 2004;Nakamura et al. 2010;Hoa et al. 2015).HR is carried out in a series of steps, beginning with the 5 0 -to-3 0 strand resection of DSBs, which is called DSB resection (reviewed in Stracker & Petrini 2011;Symington & Gautier 2011). The resulting 3 0 -overhang is coated with a single-strand DNA binding protein, replication protein A (RPA). RPA is subsequently replaced with polymerized Rad51 recombinase, which polymerization results in the formation of subnuclear Rad51 foci. Polymerized Rad51 performs homology search and strand invasion into intact homologous sequences leading to formation of D-loop and Holliday junction structures. Biochemical and genetic studies have shown that in Saccharomyces cerevisiae (S. cerevisiae), DSB resection is initiated by Mre11 nuclease, which physically associates with Rad50 and Xrs2 (the MRX complex). The MRX complex and Sae2 are the orthologs of human Mre11/ Rad50/Nbs1 (the MRN complex) and CtIP, respectively. Yeast MRX ...

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Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.

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Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5′-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3′ single-strand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51–DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells. Depletion of Mre11 decreases the efficiency of DSB resection only by 2- to 3-fold in mammalian cells. We show that although Mre11 is required for efficient HR-dependent repair of ionizing-radiation–induced DSBs, Mre11 is largely dispensable for DSB resection in both chicken DT40 and human TK6 B cell lines. Moreover, a 2- to 3-fold decrease in DSB resection has virtually no impact on the efficiency of HR. Thus, although a large number of researchers have reported the vital role of Mre11-mediated DSB resection in HR, the role may not explain the very severe defect in HR in Mre11-deficient cells, including their lethality. We here show experimental evidence for the additional roles of Mre11 in (i) elimination of chemical adducts from DSB ends for subsequent DSB repair, and (ii) maintaining HR intermediates for their proper resolution.

Tóm tắt— Bài báo này trình bày kết quả nghiên cứu xây dựng giải pháp hỗ trợ xử lý sự cố an toàn thông tin (ATTT) phục vụ tổ chức trong Chính phủ điện tử. Giải pháp đề xuất bao gồm cả quy trình xử lý sự cố ATTT và hệ thống UET.SIR phục vụ công tác hỗ trợ xử lý sự cố ATTT. Quy trình được xây dựng dựa trên kết hợp các tiêu chuẩn trong nước và quốc tế, được tuỳ biến để phù hợp với thực tiễn của chính phủ điện tử. Hệ thống UET.SIR hỗ trợ xử lý xự cố bao gồm USB chuyên dụng phục vụ thu thập chứng cứ số sự cố ATTT, và phần mềm trung tâm với các chức năng phân tích chứng cứ sự cố để phát hiện nguyên nhân và hỗ trợ công tác xử lý. Kết quả thử nghiệm hệ thống UET.SIR tại Bộ Tài nguyên và Môi trường bước đầu đã chứng minh được khả năng áp dụng thực tiễn và hỗ trợ xử lý sự cố ATTT đối với một số hệ thống trọng yếu. Abstract— This paper presents the results of research on building solutions to support information security incident handling in organizations. The proposed solution includes both the procedure for information security incident handling, and the UET.SIR system for supporting information security handling. The process is built on a combination of national and international standards, but is customized to suit e-government practices. The UET.SIR system includes a dedicated USB for collecting digital evidence of ATTT incidents and central software with evidence analysis functions to detect the cause and provide support incident handling. The test results of the UET.SIR system at the Ministry of Natural Resources and Environment have initially demonstrated its practical applicability and support for information security incident handling.

This paper presents a new effective reversible data hiding method based on pixel-value-ordering (iGePVO-K) which is improvement of a recent GePVO-K method that recently is considered as a PVO-used method having highest embedding capacity. In comparison with GePVO-K method, iGePVO-K has the following advantages. First, the embedding capacity of the new method is higher than that of GePVO-K method by using data embedding formulas reasonably and reducing the location map size. Second, for embedding data, in the new method, each pixel value is modified at most by one, while in GePVO-K method, each pixel value may be modified by two. In fact, in the GePVO-K method, the largest pixels are modified by two for embedding bits 1 and by one for bits 0. This is also true for the smallest pixels. Meanwhile, in the proposed method, the largest pixels are modified by one for embedding bits 1 and are unchanged if embedding bits 0. Therefore, the stego-image quality in proposed method is better than that in GePVO-K method. Theoretical analysis and experiment results show that the proposed method has higher embedding capacity and better stego image quality than GePVO-K method.