Keywords: comet assay, DNA repair, human biomonitoring Internship Duration: 30/11/-1 - 30/11/-1
Head of the hosting team: Solange Costa
Website: Click here
Address of the host laboratory: ISPUP Team EPIUnit and ITR Rua Alexandre Herculano, 321 4000-055 Porto Portugal
Supervisor 1: Solange CostaE-mail: solange.costa2@gmail.com Phone: +351222061820
Supervisor 2: Carla CostaE-mail: cstcosta@gmail.comPhone: +351222061820
The DNA molecule is continuously under attack by a multitude of endogenous and exogenous genotoxic insults challenging the integrity of the genome by introducing a variety of DNA lesions. The efficient repair of these lesions is of paramount importance to the maintenance of the genomic integrity. When DNA damage persists and interferes with replication or transcription, DNA repair mechanisms trigger cellular senescence or apoptosis that inactivate or eliminate damaged cells. In the absence or impairment of such repair or checkpoint mechanisms, the genomic integrity of the organism is often compromised. Loss of efficient DNA damage repair can lead to accelerated ageing phenotypes, increased cancer risk or other human syndromes and pathologies. Susceptibility to these events is often driven by the cell's response to DNA damage. Therefore, DNA repair capacity/activity is regarded as an important biomarker to be evaluated when assessing the carcinogenic risk of potentially hazardous agents. The comet-based in vitro DNA repair assay is a biochemical tool that allows the measurement of repair capacity. The assay can measure the activity of two excision repair pathways, BER or NER, taking advantage of the intermediate DNA strand breaks formed at the sites of DNA lesions during the repair process. Quantification of these breaks reflects the repair activity. In short, the assay measures the enzymatic activity of a cell extract (from a tissue or cells, e.g. placenta, lymphocytes) by incubating it with a substrate of cells (e.g. HeLa cells) containing artificially induced DNA lesions (previous treatment with specific agent with a known genotoxic action). Incision at damage sites, arising from the repair process, is detected using the comet assay. The aim of the internship is to implement the comet-based in vitro DNA repair as a tool to evaluate the DNA repair capacity in human lymphocytes. A quick introduction to the comet assay and its versions will also be given.
Comet assay; Primary cells and cell lines (human)
Hakem R. DNA-damage repair; the good, the bad, and the ugly (2008) EMBO J. 27(4):589-605. doi:10.1038/emboj.2008.15 Andrew R. Collins (2014) Measuring oxidative damage to DNA and its repair with the comet assay, Biochimica et Biophysica Acta (BBA), 1840 (2): 794-800, https://doi.org/10.1016/j.bbagen.2013.04.022 Vodenkova, S., Azqueta, A., Collins, A. et al. (2020) An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity. Nat Protoc 15, 3844–3878 https://doi.org/10.1038/s41596-020-0401-x Azqueta A, Slyskova J, Langie SA, et al. (2014) Comet assay to measure DNA repair: approach and applications. Front Genet. 5:288. doi:10.3389/fgene.201
