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A rapid, safe, and quantitative in vitro assay for measurement of uracil-DNA glycosylase activity

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Abstract

Base excision repair (BER) is a frontline repair mechanism that operates through the G1 phase of the cell cycle, which ensures the genome integrity by repairing thousands of DNA lesions due to endogenous and exogenous agents. Its correct functioning is fundamental for cell viability and the health of the organism. Uracil is one of the most prevalent lesions that appears in DNA arising by spontaneous or enzymatic deamination of cytosine or misincorporation of the deoxyuridine 5′-triphosphate nucleotide (dUTP) in place of deoxythymidine 5′-triphosphate (dTTP) during DNA replication. In the first pathway, the uracil will preferentially pair with adenine, leading to C:G → T:A transition. When uracil in DNA arises from misincorporation of dUTP instead of dTTP, this process will result in A:U pairs. Organisms counteract the mutagenic effects of uracil in DNA using the BER repair system, which is mediated by a member of the uracil-DNA glycosylase (UDG) superfamily. Several assays evaluating the in vitro BER enzyme activity have been described so far. Some of these measure the BER activity by an oligonucleotide incision assay using radiolabeled duplex oligo. Others use circular double-stranded DNA substrates containing a defined lesion. The novelty of our method resides in its rapidity and safety (radioactive free detection) as well as in the possibility of having a reliable quantitative determination of UDG activity in both cell and tissue extracts. We also demonstrated the effectiveness of our method in assessing UDG activity in cell lines with a reduced DNA repair capacity and in different kinds of tissues.

Key messages

• Base excision repair is a fundamental repair mechanism ensuring the genome integrity.

• Uracil is one of the most prevalent lesions that appears in DNA.

• The mutagenic effects of uracil in DNA are mitigated by the uracil-DNA glycosylase.

• Several assays evaluating the in vitro BER activity have been described so far.

• A safe and quantitative assay evaluating the in vitro UDG activity is required.

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Abbreviations

BER:

Base excision repair

dUTP:

Deoxyuridine 5′-triphosphate nucleotide

dTTP:

Deoxythymidine 5′-triphosphate

UDG:

Uracil-DNA glycosylase

AP:

Apurinic/apyrimidinic

HhH:

Helix-hairpin-helix

MPG:

3-Methyl-purine glycosylase

NEIL:

Endonuclease VIII-like

MSCs:

Mesenchymal stromal cells

UNG:

Uracil N-glycosylase

SMUG1:

Single-strand-selective mono-functional uracil-DNA glycosylase 1

TDG:

Thymine DNA glycosylase

MBD4:

Methyl-CpG-binding domain 4

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Acknowledgements

M.A.B.M. and T.S. acknowledge PON I&C 2014-2020 “Micro/nanoformulati innovativi per la valorizzazione di molecole bioattive, utili per la salute e il benessere delle popolazione, ottenute da prodotti di scarto della filiera ittica (FOR.TUNA)” project, Grant/Award Number: F/050347/03IX32 – Ministero dello Sviluppo Economico (MiSE).

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Author notes

  1. Tiziana Squillaro and Mauro Finicelli contributed equally to this work.

Authors and Affiliations

  1. Department of Medical, Surgical, Neurological, Metabolic Sciences, and Aging, 2nd Division of Neurology, Center for Rare Diseases and InterUniversity Center for Research in Neurosciences, University of Campania “Luigi Vanvitelli”, Naples, Italy

    Tiziana Squillaro & Mariarosa Anna Beatrice Melone

  2. Research Institute on Terrestrial Ecosystems, National Research Council, Naples, Italy

    Mauro Finicelli & Gianfranco Peluso

  3. Department of Experimental Medicine, University of Campania “Luigi Vanvitelli”, Naples, Italy

    Nicola Alessio, Stefania Del Gaudio, Giovanni Di Bernardo & Umberto Galderisi

  4. Sbarro Institute for Cancer Research and Molecular Medicine, Department of Biology, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA

    Mariarosa Anna Beatrice Melone & Umberto Galderisi

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  1. Tiziana Squillaro
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  2. Mauro Finicelli
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  3. Nicola Alessio
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  4. Stefania Del Gaudio
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Contributions

TS and MF planned and performed experiments and wrote the paper; SDG performed experiments; NA and GDB analyzed data; MABM and GP contributed reagents and other essential material; UG conceived and supervised the study.

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Correspondence to Umberto Galderisi.

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Squillaro, T., Finicelli, M., Alessio, N. et al. A rapid, safe, and quantitative in vitro assay for measurement of uracil-DNA glycosylase activity. J Mol Med 97, 991–1001 (2019). https://doi.org/10.1007/s00109-019-01788-8

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  • DOI: https://doi.org/10.1007/s00109-019-01788-8

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