Formamidopyrimidine—DNA Glycosylase Targeted to Specific Organelles in C2C12 Cells

Summary: Targeting mitochondria or nucleus with an oxidised DNA base remover

Interestingness: 3

Paper by Karah A Street, Kerrie L. Hall, Patrick Murphy and Christi A Walter in the Journal of Anti-Aging Medicine, Volume 2, Issue 3, Fall 1999.

(((
The follow up paper to this one could be very interesting. This one seems to show that they could target either the mitochondria, or the nucleus with a protein, formamidopyrimidine-DNA glycosylase (Fpg), that gets rid of 2-deoxy-8-hydroxyguanine (8-OHdG), a screwed up version of the guanine base and the most common oxidised base. 8-OHdG causes the guanines (G) to be replaced by thymine (T) (by the normal repair mechanism I think). Fpg gets rid of 8-OHdG by taking out the base and leaving the ribose chain. This is supposedly a part of one of the normal DNA fixing mechanisms, called the base excision repair (BER), where one protein gets rid of a mutated base, and another goes and inserts the right base in.

So, yes, they created two DNA vectors, inserted them into some mouse muscle cells, and mostly saw what they were looking for, with the nuclear DNA being expressed mostly in the nucleus, and the mitochondrial in the cytoplasm. The levels of the molecule seemed pretty low though, and didn't correlate with the number of copies they inserted.

No assessment of the amount of 8-OHdG damage in the DNAs after transfection was done. I assume that's part of the plan for future work.
)))


Abstract follows:

Mitochondrial respiration provides a major source of energy for eukaryotic cells. However, the energy-producing processes also generate reactive oxygen species, which in turn damage mitochondrial DNA found in the mitochondrial matrix. Due to its locale, mitochondrial DNA is more susceptible to oxidative damage than nuclear DNA. While mitochondria do have some DNA repair capabilities, particularly base excision repair, oxidative damage persists in mitochondrial DNA. Correlations have been demonstrated between increasing age and increased levels of oxidative damage and mitochondrial DNA mutations. The current experiments were designed to begin to more directly delineate the role oxidative damage in mitochondrial DNA plays in aging. The mouse myoblast cell line, C2C12, was transfected with vectors, which express formamidopyrimidine-DNA glycosylase-myc fusion protein (Fpg-myc) and which contain either a mitochondrial or nuclear localization signal. Positive transfectants display expression of fpg at the mRNA level and exhibit an increase in Fpg activity in a whole-cell protein extract using a Fpg activity assay. Immunofluorescence analyses confirm that the transfected vectors have Fpg-myc appropriately targeted to mitochondria or nuclei. These cell lines with specifically targeted Fpg-myc expression provide the tools to test the effects of increasing the levels of a DNA glycosylase in mitochondria and nuclei on oxidative damage in DNA.