Mutation in the Glucose-6-phosphate Dehydrogenase Gene Leads to Inactivation of Ku DNA End Binding during Oxidative Stress

Ayene, Iraimoudi S.; Stamato, Thomas D.; Mauldin, Stanley K.; Biaglow, John E.; Tuttle, Steven W.; Jenkins, Susan F.; Koch, Cameron J.

doi:10.1074/jbc.m111366200

Cited by 46 publications

(51 citation statements)

References 48 publications

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“…Ku heterodimer contains 14 cysteine residues and 5 of these are located in Ku70, which is in contact with the DNA binding site (22). In a recent report, we have shown that Ku protein function can be greatly decreased in glucose-6-phosphate dehydrogenase -deficient Chinese hamster ovary cells by Ku protein thiol oxidation (23). However, this biochemical approach is effective only in glucose-6-phosphate dehydrogenase -deficient cells.…”

Section: Introductionmentioning

confidence: 99%

Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and γ radiation

Ayene

Ford²,

Koch

2005

Molecular Cancer Therapeutics

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Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiationresponsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to ; radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to ; radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments. [Mol Cancer Ther 2005;4(4):529 -36]

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Section: Introductionmentioning

confidence: 99%

Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and γ radiation

Ayene

Ford²,

Koch

2005

Molecular Cancer Therapeutics

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“…2 TrxR activity is regulated by NADPH produced with glucose-6-phosphate dehydrogenase (G6PD), which is the allosteric enzyme of oxidative reactions of the pentose phosphate pathway. 3 Three types of thioredoxin reductase enzymes were determined in mammals: TrxR1 found in the cell cytoplasm, 4 TrxR2 found in the mitochondria, 5,6 and TrxR3 found in the testicles. 7 Mainly two TrxRs were cloned: an enzyme of 54 kDa found in the cytoplasm and an enzyme of 56 kDa found in the mitochondria.…”

Section: Introductionmentioning

confidence: 99%

Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity

Temel¹,

Çiftçi²

2017

Turk J Chem

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Abstract:The thioredoxin system, found in all living creatures, consists of thioredoxin protein (Trx), thioredoxin reductase enzyme (TrxR), and NADPH. In this study, turkey liver mitochondrial TrxR enzyme with 3.07 EU × mg −1 specific activity was purified 990-fold in a yield of 2.05% using 2',5'-ADP Sepharose 4B affinity chromatography. The purity of the enzyme was measured and the molecular weight of its subunits was determined to be 45.5 kDa by SDS-PAGE. The molecular mass of the enzyme's natural state was found to be 88 kDa using Sephadex-G 150 gel filtration chromatography. In addition, characteristic and kinetic properties of the enzyme were determined. ion increased enzyme activity, and Ni 2+ and Co 2+ ions had no effect on enzyme activity.

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“…Regulation of redox stress via genetic mutation has also been found to influence DNA DSB repair (21). Analysis of the Ku protein reveals a decrease in DNA-binding activity as a function of oxidative stress (22).…”

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confidence: 99%

Kinetic Analysis of the Ku-DNA Binding Activity Reveals a Redox-dependent Alteration in Protein Structure That Stimulates Dissociation of the Ku-DNA Complex

Andrews

Lehman

Turchi

2006

Journal of Biological Chemistry

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Ku is a heterodimeric protein comprising 70-and 80-kDa subunits that participate in the non-homologous end-joining (NHEJ) repair pathway for rejoining DNA double strand breaks. We have analyzed the pre-steady state binding of Ku with various DNA duplex substrates and identified a redox-sensitive Ku-DNA interaction. Pre-steady state analysis of Ku DNA binding was monitored via intrinsic Ku quenching upon binding DNA and revealed that, under fully reduced conditions, binding occurred in a single-step process. Reactions performed under limited reduction revealed a two-step binding process, whereas under fully oxidized conditions, we were unable to detect quenching of Ku fluorescence upon binding DNA. The differential quenching observed under the different redox conditions could not be attributed to two Ku molecules binding to a single substrate or Ku sliding inward on the substrate. Although only modest differences in Ku DNA binding activity were observed in the stoichiometric anisotropy and electrophoretic mobility shift assay studies, as a function of redox conditions, a dramatic difference in the rate of Ku dissociation from DNA was observed. This effect was also induced by diamide treatment of Ku and could be abrogated by dithiothreitol treatment, demonstrating a reversible redox effect on the stability of the Ku-DNA complex. The redox-dependent alteration in Ku-DNA interactions is manifested by a redox-dependent alteration in Ku structure, which was confirmed by limited proteolysis and mass spectrometry analyses. The results support a model for the interaction of Ku with DNA that is regulated by redox status and is achieved by altering the dissociation of the Ku-DNA complex. DNA double strand breaks (DSBs)2 can be a lethal type of DNA damage. Cells have developed two major pathways in which to remove such damage, homologous recombination and NHEJ (1). The Ku autoantigen is an essential component of the NHEJ pathway. Ku shows a high affinity for double strand DNA termini (2), and once bound to the DNA end, increases the affinity of the DNA-PK catalytic subunit (DNA-PKcs) for the DNA termini (3). To accommodate the large 469-kDa DNA-PKcs, Ku translocates inward on the DNA in an ATP-independent manner, ultimately forming the active DNA-PK complex (3, 4). Activated DNA-PK then phosphorylates target proteins, including Artemis (5), Mre11/Rad50/NBS1 complex (6), and DNA ligase IV/XRCC4 (7), all of which participate in either signaling events in response to DNA damage or completing the repair process.The crystal structure of free Ku and the Ku-DNA complex has been solved and reveals a novel DNA binding structure comprising a base, two pillars, and a bridge through which the duplex DNA can thread (8). The Ku70 and -80 subunits share minimal sequence homology, yet adopt similar three-dimensional structures, with each subunit contributing to the base, pillars, and bridge (8). This ring-like structure of Ku provides the structural basis for the DNA termini requirement for binding as well as the energy-independent ability...

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Mutation in the Glucose-6-phosphate Dehydrogenase Gene Leads to Inactivation of Ku DNA End Binding during Oxidative Stress

Cited by 46 publications

References 48 publications

Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and γ radiation

Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and γ radiation

Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity

Kinetic Analysis of the Ku-DNA Binding Activity Reveals a Redox-dependent Alteration in Protein Structure That Stimulates Dissociation of the Ku-DNA Complex

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