Time-resolved chloroquine-induced relaxation of supercoiled plasmid DNA

Mahut, Marek; Leitner, Michael; Ebner, Andreas; Lämmerhofer, Michael; Hinterdorfer, Peter; Lindner, Wolfgang

doi:10.1007/s00216-011-5213-y

Cited by 9 publications

(9 citation statements)

References 28 publications

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“…APDMES or APTES. Similar findings were observed also by other authors [34][35][36][37] , describing presence of clearly non-DNA objects on the surface of silanized mica, however, non-APS silanization was used (mainly APTES based) [38][39][40] . The fractions of APS compound used for mica silanization are inserted in the AFM images as labels.…”

Section: Roughness Analysissupporting

confidence: 88%

Improved method for Mica functionalization used in single molecule imaging of DNA with atomic force microscopy

et al. 2016

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The modified procedure of 1-(3-aminopropyl)silatrane (APS) compound synthesis based on a new derivative (3-aminopropyl)trimethoxysilane for the purpose of DNA immobilization for AFM single imaging is described. New reaction pathway based on kinetically driven reaction approach is described. Necessity of two-step purification process is proved; ability of purified APS to provide four times smoother surfaces in comparison with a crude product is demonstrated. Various analytical methods such mass spectroscopy and 1 H NMR were used to show structure and enhanced purity of the APS product. APS mediates fixation of DNA molecules to mica substrates to be used for DNA imaging under Atomic Force Microscope. The use of an APS compound for simple and rapid silanization of mica surface is demonstrated. The advantages of APS-based method are based mainly on low roughness of modified mica and homogeneous surface coverage by short sequence dsDNA (246 bp). The product obtained by the condensation reaction was purified in a two step process whose effectiveness was demonstrated not only by reduction of the silanized surface roughness, but also by mass spectroscopy (MS-ESi), MALDI-TOF method and proton magnetic resonance spectroscopy. Experiments demonstrate that 1-(3-aminopropyl)silatrane can be used to fix dsDNA molecules to a mica surface to be visualized by either the tapping mode or the force-volume mode of AFM microscopy, as demonstrated by experiments. Moreover, necessity of advanced purification protocol is demonstrated by AFM based roughness measurementspure vs crude APS product. The kinetics of APS-layer aging, caused by silicon oxide growth on silanized layers, was studied by water contact angle measurements and is discussed.

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Section: Roughness Analysissupporting

confidence: 88%

Improved method for Mica functionalization used in single molecule imaging of DNA with atomic force microscopy

et al. 2016

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“…To test this possibility, we injected 30 μl of 93.8–156.3 μM chloroquine, which can alter chromatin and chromosome structures (54), into 6-week-old mouse testes and studied the effects 3 weeks later. Injection of 156.3 μM chloroquine clearly destroyed the spermatogenesis process in Rnf20 Flox/Flox mice, while the lower dose of 93.8 μM chloroquine did not result in any observable effect on Rnf20 Flox/Flox mouse testes (Figure 6A and Supplementary Figure S13A).…”

Section: Resultsmentioning

confidence: 99%

“…Most importantly, we found that the meiotic recombination and spermatogenesis defects of Stra8-Rnf20 −/− mice were partially rescued by the injection of a 4-aminoquinoline drug that has long been used in the treatment or prevention of malaria (71). Chloroquine is also a chromatin relaxation agent that can intercalate into the nuclear strands and force chromatin relaxation (54). During meiotic recombination in Stra8-Rnf20 −/− spermatocytes or H2BK123R yeast cells, the insertion of chloroquine into meiotic chromatin might relax the chromatin, which can mimic the function of H2B ubiquitination, thus partially restoring the meiotic recombination process (Figure 8C).…”

Section: Discussionmentioning

confidence: 99%

H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation

Song

et al. 2016

Nucleic Acids Res

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Meiotic recombination is essential for fertility in most sexually reproducing species, but the molecular mechanisms underlying this process remain poorly understood in mammals. Here, we show that RNF20-mediated H2B ubiquitination is required for meiotic recombination. A germ cell-specific knockout of the H2B ubiquitination E3 ligase RNF20 results in complete male infertility. The Stra8-Rnf20−/− spermatocytes arrest at the pachytene stage because of impaired programmed double-strand break (DSB) repair. Further investigations reveal that the depletion of RNF20 in the germ cells affects chromatin relaxation, thus preventing programmed DSB repair factors from being recruited to proper positions on the chromatin. The gametogenetic defects of the H2B ubiquitination deficient cells could be partially rescued by forced chromatin relaxation. Taken together, our results demonstrate that RNF20/Bre1p-mediated H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation, and suggest an old drug may provide a new way to treat some oligo- or azoospermia patients with chromatin relaxation disorders.

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“…CQ is a well-known DNA intercalating drug that can unwind the DNA double helix, altering the conformation of DNA [22, 23]. Therefore, we hypothesized that with its oxidative stress-inducing property, CQ can induce DNA damage.…”

Section: Resultsmentioning

confidence: 99%

The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair

et al. 2016

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Triple negative breast cancer (TNBC), characterized by an abundance of treatment-resistant breast cancer stem cells (CSCs), has a poorer prognosis than other types of breast cancers. Despite its aggressiveness, no effective targeted therapy exists for TNBC. Here, we demonstrate that CQ effectively targets CSCs via autophagy inhibition, mitochondrial structural damage, and impairment of double-stranded DNA break repair. Electron microscopy demonstrates CQ-induced mitochondrial cristae damage, which leads to mitochondrial membrane depolarization with a significant reduction in the activity of cytochrome c oxidase and accumulation of superoxide and double-stranded DNA breaks. CQ effectively diminishes the TNBC cells’ ability to metastasize in vitro and in a TNBC xenograft model. When administered in combination with carboplatin, CQ effectively inhibits carboplatin-induced autophagy. This combination treatment significantly diminishes the expression of DNA repair proteins in CSC subpopulations, resulting in tumor growth reduction in carboplatin-resistant BRCA1 wild-type TNBC orthotopic xenografts. As TNBC’s high treatment failure rate has been attributed to enrichment of CSCs, CQ, an autophagy inhibitor with anti-CSC effects, may be an effective adjunct to current TNBC chemotherapy regimens with carboplatin.

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Time-resolved chloroquine-induced relaxation of supercoiled plasmid DNA

Cited by 9 publications

References 28 publications

Improved method for Mica functionalization used in single molecule imaging of DNA with atomic force microscopy

Improved method for Mica functionalization used in single molecule imaging of DNA with atomic force microscopy

H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation

The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair

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