Role of endonuclease G in exogenous DNA stability in HeLa cells

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.

Unleashing a novel function of Endonuclease G in mitochondrial genome instability

“…Considering that Endonuclease G is one of the essential mitochondrial nucleases ( McDermott-Roe et al, 2011 ; Misic et al, 2016 ; Wiehe et al, 2018 ; Zhou et al, 2016 ), the presence of Endonuclease G was examined in mitochondria, using mitochondrial specific marker, ‘MitoTracker Deep Red or MitoTracker green FM’ and using immunofluorescence with Endonuclease G antibody in three cell lines of different origin ( Figure 8A ; Figure 8—figure supplement 1 ). Merge of MitoTracker with tagged Endonuclease G revealed localization of Endonuclease G in mitochondria in HeLa (human cervical cancer), HEK 293T (human embryonic kidney epithelial) and MEF (mouse embryonic fibroblast) cell lines ( Figure 8A and B ; Figure 8—figure supplement 1 ).…”

Unleashing a Novel Function of Endonuclease G in Mitochondrial Genome Instability

“…Considering that Endonuclease G is one of the important mitochondrial nucleases (Gole et al, 2015;McDermott-Roe et al, 2011;Misic et al, 2016;Wiehe et al, 2018;Zhou et al, 2016), its presence was confirmed in mitochondria, using mitochondrial specific marker, 'MitoTracker Deep Red or MitoTracker green FM' and using immunofluorescence with Endonuclease G antibody in three different cell lines of different origin (Figure 3G; S4A). Merge of MitoTracker with tagged Endonuclease G revealed localization of Endonuclease G in mitochondria in HeLa (human cervical cancer), HEK 293T (human embryonic kidney epithelial) and MEF (mouse embryonic fibroblast) cell lines (Figure 3G; S4A, B).…”