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Role of endonuclease G in exogenous DNA stability in HeLa cells

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Abstract

Endonuclease G (EndoG) is a well-conserved mitochondrial-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four-day time course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances, it may nonspecifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from nonviral DNA vectors in gene therapy efforts.

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Abbreviations

CMVie:

cytomegalovirus immediate/early

EndoG:

endonuclease G

GFP:

green fluorescent protein

MCS:

multi-cloning site

MLS:

mitochondria localization signal

ORF:

open reading frame

PBS:

phosphate buffered saline

PTE:

primary tubular epithelial (cells)

qPCR:

quantitative PCR

ROS:

reactive oxygen species

RT:

reverse transcription

shRNA:

short-hairpin RNA

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Authors and Affiliations

  1. Department of Biological Sciences, Brock University, 500 Glenridge Avenue, St. Catharines, ON, L2S 3A1, Canada

    V. Misic, M. El-Mogy & Y. Haj-Ahmad

  2. Molecular Biology Department, National Research Centre, 33 El-Bohouth St., Dokki, Giza, 12622, Egypt

    M. El-Mogy

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  1. V. Misic
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Published in Russian in Biokhimiya, 2016, Vol. 81, No. 2, pp. 265–281.

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Misic, V., El-Mogy, M. & Haj-Ahmad, Y. Role of endonuclease G in exogenous DNA stability in HeLa cells. Biochemistry Moscow 81, 163–175 (2016). https://doi.org/10.1134/S0006297916020103

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