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      Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

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          Abstract

          Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population.

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          Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function.

          Satya Prakash, Robert Johnson, Louise Prakash (2005)
          This review focuses on eukaryotic translesion synthesis (TLS) DNA polymerases, and the emphasis is on Saccharomyces cerevisiae and human Y-family polymerases (Pols) eta, iota, kappa, and Rev1, as well as on Polzeta, which is a member of the B-family polymerases. The fidelity, mismatch extension ability, and lesion bypass efficiencies of these different polymerases are examined and evaluated in the context of their structures. One major conclusion is that, despite the overall similarity of basic structural features among the Y-family polymerases, there is a high degree of specificity in their lesion bypass properties. Some are able to bypass a particular DNA lesion, whereas others are efficient at only the insertion step or the extension step of lesion bypass. This functional divergence is related to the differences in their structures. Polzeta is a highly specialized polymerase specifically adapted for extending primer termini opposite from a diverse array of DNA lesions, and depending upon the DNA lesion, it contributes to lesion bypass in a mutagenic or in an error-free manner. Proliferating cell nuclear antigen (PCNA) provides the central scaffold to which TLS polymerases bind for access to the replication ensemble stalled at a lesion site, and Rad6-Rad18-dependent protein ubiquitination is important for polymerase exchange.
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            The comet assay: topical issues.

            Rudolf Stetina, Amaia Azqueta Oscoz, Samuel R A Collins … (2008)
            The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo-or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.
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              Eukaryotic DNA replication origins: many choices for appropriate answers.

              Marcel Mechali (2010)
              At each cell division in humans, 30,000-50,000 DNA replication origins are activated, and it remains unclear how they are selected and recognized by replication factors. DNA replication in multicellular organisms must accommodate variations in growth conditions and DNA damage. It must also adapt to changes in chromatin organization associated with cell differentiation and development. The selection of replication origins in metazoans seems to involve multiple choices, with the appropriate answers depending on the identity of the cell or the conditions of growth. This suggests that during evolution, the use of replication origins became more controlled by epigenetic mechanisms affecting chromosome dynamics and expression than by DNA synthesis per se.
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                Author and article information

                Contributors
                Michael R. Volkert: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2013
                Publication date (Electronic): 6 August 2013
                Volume: 8
                Issue: 8
                Electronic Location Identifier: e70391
                Affiliations
                [1 ]Institute of Genetics, Biological Research Centre of Hungarian Academy of Sciences, Szeged, Hungary
                [2 ]Biomedical Sciences Research Institute, School of Biomedical Sciences, University of Ulster, Coleraine, Londonderry, Northern Ireland
                University of Massachusetts Medical School, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MM IR CSD LH. Performed the experiments: MM HG. Analyzed the data: MM HG LH. Wrote the paper: MM HG LH.

                [¤]

                Current address: Institute of Genetics, Biological Research Centre of Hungarian Academy of Sciences, Szeged, Hungary

                Article
                Publisher ID: PONE-D-13-12956
                DOI: 10.1371/journal.pone.0070391
                PMC ID: 3735594
                PubMed ID: 23936422
                SO-VID: 056009e3-b01f-4e52-b507-75d371a3ee1c
                Copyright statement: Copyright @ 2013
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 27 March 2013
                Date accepted : 19 June 2013
                Page count
                Pages: 9
                Funding
                This work was supported by the Hungarian Science Foundation grants (OTKA 101225, GOP-1.1.1-11-2012-0030 and GOP-1.1.1-11-2012-0384), and IPA Cross-border Co-operation Programme (HUSRB/1002/214/126). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Subject: Biology
                Subject: Biochemistry
                Subject: Nucleic Acids
                Subject: DNA
                Subject: DNA repair
                Subject: DNA replication
                Subject: Biophysics
                Subject: Nucleic Acids
                Subject: DNA
                Subject: DNA repair
                Subject: DNA replication
                Subject: Molecular Cell Biology
                Subject: Nucleic Acids
                Subject: DNA
                Subject: DNA repair

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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