Next generation high throughput DNA damage detection platform for genotoxic compound screening

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Abstract

Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.

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Most cited references 54

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Single-strand break repair and genetic disease.

Keith Caldecott (2008)

Hereditary defects in the repair of DNA damage are implicated in a variety of diseases, many of which are typified by neurological dysfunction and/or increased genetic instability and cancer. Of the different types of DNA damage that arise in cells, single-strand breaks (SSBs) are the most common, arising at a frequency of tens of thousands per cell per day from direct attack by intracellular metabolites and from spontaneous DNA decay. Here, the molecular mechanisms and organization of the DNA-repair pathways that remove SSBs are reviewed and the connection between defects in these pathways and hereditary neurodegenerative disease are discussed.

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Requirement of mammalian DNA polymerase-beta in base-excision repair.

R W Sobol, J Horton, R. Kuhn … (1996)

Synthesis of DNA by DNA polymerase-beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of beta-polymerase in different types of DNA repair. However, the significance of these studies to the in vivo role of beta-polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA polymerase-beta. Extracts from these cell lines were found to be defective in uracil-initiated base-excision repair. The beta-polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-excision repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type beta-polymerase minitransgene. These studies demonstrate that beta-polymerase functions specifically in base-excision repair in vivo.

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Methylating agents and DNA repair responses: Methylated bases and sources of strand breaks.

Douglas Pittman, Jeffrey D. Wyatt (2006)

The chemical methylating agents methylmethane sulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) have been used for decades as classical DNA damaging agents. These agents have been utilized to uncover and explore pathways of DNA repair, DNA damage response, and mutagenesis. MMS and MNNG modify DNA by adding methyl groups to a number of nucleophilic sites on the DNA bases, although MNNG produces a greater percentage of O-methyl adducts. There has been substantial progress elucidating direct reversal proteins that remove methyl groups and base excision repair (BER), which removes and replaces methylated bases. Direct reversal proteins and BER, thus, counteract the toxic, mutagenic, and clastogenic effects of methylating agents. Despite recent progress, the complexity of DNA damage responses to methylating agents is still being discovered. In particular, there is growing understanding of pathways such as homologous recombination, lesion bypass, and mismatch repair that react when the response of direct reversal proteins and BER is insufficient. Furthermore, the importance of proper balance within the steps in BER has been uncovered with the knowledge that DNA structural intermediates during BER are deleterious. A number of issues complicate the elucidation of the downstream responses when direct reversal is insufficient or BER is imbalanced. These include inter-species differences, cell-type-specific differences within mammals and between cancer cell lines, and the type of methyl damage or BER intermediate encountered. MMS also carries a misleading reputation of being a radiomimetic, that is, capable of directly producing strand breaks. This review focuses on the DNA methyl damage caused by MMS and MNNG for each site of potential methylation to summarize what is known about the repair of such damage and the downstream responses and consequences if the damage is not repaired.

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Author and article information

Contributors

Robert W. Sobol:

ORCID: http://orcid.org/0000-0001-7385-3563

rwsobol@health.southalabama.edu

Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group UK (London )

ISSN (Electronic): 2045-2322

Publication date (Electronic): 9 February 2018

Publication date PMC-release: 9 February 2018

Publication date Collection: 2018

Volume: 8

Electronic Location Identifier: 2771

Affiliations

[1 ]ISNI 0000 0000 9552 1255, GRID grid.267153.4, Department of Oncologic Sciences, Mitchell Cancer Institute, , University of South Alabama, ; 1660 Springhill Avenue, Mobile, AL 36604 USA

[2 ]ISNI 0000 0001 2110 5790, GRID grid.280664.e, Division of the National Toxicology Program, , National Institute of Environmental Health Sciences, ; Research Triangle Park, NC 27709, USA

[3 ]DGL-Imaging, Millersville, MD 21108 USA

[4 ]ISNI 0000 0001 2341 2786, GRID grid.116068.8, Department of Biological Engineering, MIT, ; Cambridge, MA 02139 USA

Author information

Robert W. Sobol http://orcid.org/0000-0001-7385-3563

Article

Publisher ID: 20995

DOI: 10.1038/s41598-018-20995-w

PMC ID: 5807538

PubMed ID: 29426857

SO-VID: b26493ec-bc98-4fe0-9f1c-2e4e4f2fab23

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Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 5 September 2017

Date accepted : 29 January 2018

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Next generation high throughput DNA damage detection platform for genotoxic compound screening

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Most cited references 54

Single-strand break repair and genetic disease.

Requirement of mammalian DNA polymerase-beta in base-excision repair.

Methylating agents and DNA repair responses: Methylated bases and sources of strand breaks.

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