Analysis of the Action of the Restriction Endonuclease AluI Using Three Different Comet Assay Protocols

Müller, Wolfgang‐Ulrich; Ciborovius, Jens; Bauch, Thomas; Johannes, Christian; Schunck, Christian H.; Mallek, Ulrike; Böcker, Wilfried; Obe, Günter; Streffer, C.

doi:10.1007/s00066-004-1255-9

Cited by 9 publications

(4 citation statements)

References 30 publications

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“…The use of commercial restriction enzymes (primarily AluI and PvuII) has been used for a long time in research in DNA damage and radiation biology. In these articles they clearly show that the restriction enzymes (introduced in the cytoplasm by electroporation) enters the nucleus and are solid responsible for double strand DNA breaks and chromosomal aberrations to CHO cells [28] – [30] . Hence, the presence of REs in the nucleus after microinjection of the bacterial lysate is reasonable to assume.…”

Section: Discussionmentioning

confidence: 99%

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

et al. 2014

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The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

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Section: Discussionmentioning

confidence: 99%

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

et al. 2014

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“…Approaches to measure radiosensitivity include single cell assays: comet assay [4,12,25], S-phase delay, or MNT. MN derive from chromosome fragments arising from unrepaired DNA double-strand breaks or they represent whole chromosomes that are not incorporated into the nucleus at cell division.…”

Section: Discussionmentioning

confidence: 99%

Increased Radiosensitivity as an Indicator of Genes Conferring Breast Cancer Susceptibility

et al. 2007

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The MNT detects a cellular phenotype common to a portion of sporadic breast cancer patients. This phenotype is very likely to be genetically determined. For the genetic dissection of breast cancer susceptibility this phenotype may turn out to be more efficient than breast cancer itself. Additional parameters which can be measured simultaneously with the MN frequency may be able to further enhance its usefulness.

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“…For the neutral comet assay, a modified protocol of Mueller et al (32) was applied. Cells were embedded in agarose as above and then submersed for 15 min at 18 °C in lysis buffer at pH 9.5 (87 mmol/l sodiumdodecyl sulfate (SDS), 34 mmol/l lauroylsarcosine, 25 mmol/l EDTA; Roth).…”

Section: Comet Assaymentioning

confidence: 99%

Sodium-iodide symporter positive cells after intracellular uptake of 99mTc versus α-emitter 211At

Kotzerke¹,

Wendisch²,

Freudenberg³

et al. 2012

Nuklearmedizin

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Analysis of the Action of the Restriction Endonuclease AluI Using Three Different Comet Assay Protocols

Abstract: Neither the strictly alkaline nor the strictly neutral comet assay is applicable in the radiation dose range of about 2 Gy. The restriction enzyme results show that other factors than just DNA strand breaks contribute to DNA migration into the tail of the comets.

Cited by 9 publications

References 30 publications

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

Increased Radiosensitivity as an Indicator of Genes Conferring Breast Cancer Susceptibility

Sodium-iodide symporter positive cells after intracellular uptake of 99mTc versus α-emitter 211At

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