Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay
Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2014-07-01
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| Series: | Frontiers in Genetics |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fgene.2014.00232/full |
| _version_ | 1818422872747540480 |
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| author | Jason Luke Parsons Catherine Marie Nickson |
| author_facet | Jason Luke Parsons Catherine Marie Nickson |
| author_sort | Jason Luke Parsons |
| collection | DOAJ |
| description | Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionising radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (~20-50 %) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumour suppressor protein ARF and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A-DDB1-STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. |
| first_indexed | 2024-12-14T13:33:10Z |
| format | Article |
| id | doaj.art-796ef0d1001a41ed80cb6063c2557839 |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-12-14T13:33:10Z |
| publishDate | 2014-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-796ef0d1001a41ed80cb6063c25578392022-12-21T22:59:38ZengFrontiers Media S.A.Frontiers in Genetics1664-80212014-07-01510.3389/fgene.2014.0023298174Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assayJason Luke Parsons0Catherine Marie Nickson1University of LiverpoolUniversity of LiverpoolBase excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionising radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (~20-50 %) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumour suppressor protein ARF and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A-DDB1-STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity.http://journal.frontiersin.org/Journal/10.3389/fgene.2014.00232/fullComet AssayDNA DamageDNA RepairUbiquitinubiquitylationBase excision repair |
| spellingShingle | Jason Luke Parsons Catherine Marie Nickson Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay Frontiers in Genetics Comet Assay DNA Damage DNA Repair Ubiquitin ubiquitylation Base excision repair |
| title | Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay |
| title_full | Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay |
| title_fullStr | Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay |
| title_full_unstemmed | Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay |
| title_short | Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay |
| title_sort | monitoring regulation of dna repair activities of cultured cells in gel using the comet assay |
| topic | Comet Assay DNA Damage DNA Repair Ubiquitin ubiquitylation Base excision repair |
| url | http://journal.frontiersin.org/Journal/10.3389/fgene.2014.00232/full |
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