Mitochondrial inhibition of uracil-DNA glycosylase is not mutagenic

Abstract

Background: Uracil DNA glycosylase (UDG) plays a major role in repair of uracil formed due to deamination of cytosine. UDG in human cells is present in both the nucleus and mitochondrial compartments. Although, UDG's role in the nucleus is well established its role in mitochondria is less clear.

Results: In order to identify UDG's role in the mitochondria we expressed UGI (uracil glycosylase inhibitor) a natural inhibitor of UDG in the mitochondria. Our studies suggest that inhibition of UDG by UGI in the mitochondria does not lead to either spontaneous or induced mutations in mtDNA. Our studies also suggest that UGI expression has no affect on cellular growth or cytochrome c-oxidase activity.

Conclusions: These results suggest that human cell mitochondria contain alternatives glycosylase (s) that may function as back up DNA repair protein (s) that repair uracil in the mitochondria.

Figure 1

PCR screening for stable integrants of pTZUGI in MCF12A cells. PCR using pTZUGI primers were used to screen for stable integrants. Lane 1 is a positive control (pTZUgi plasmid DNA), lane 2, 3, 4, 5, 7, 8, 9 and 10 show the presence of stable integrants.

Figure 2

RT PCR to verify expression of Ugi gene transfected in MCF12A cells using primers specic to the UGI gene: RT PCR products electrophoresed on a 1% agarose gel. Lane 1 shows RT PCR product from MCF12A cell line, lane 2 shows RT PCR product from MCF12A cells transfected with pCMV UNG, lane 3 shows RT PCR product from MCF12A transfected with empty pCMV/myc/mito control vector, lane 4 shows RT PCR product from MCF12A transfected with pCMV UGI vector.

Figure 3

Western blot analysis of mitochondrial UDG expression in transfected cell lines: Upper panel shows western blotting of mitochondrial extracts with UDG antibody the lower panel shows the same blot probed with Cox II antibody to assess for equal loading of the samples. Lane 1 is mitochondrial extract from parental MCF12A cells, lane 2 is mitochondrial extract from MCF12A cells transfected with empty pCMV/myc/mito vector, lane 3 is mitochondrial extract from MCF12A cells transfected with pCMV UNG vector, lane 4 is mitochondrial extract from MCF12A cells transfected with pCMV UGI vector. A band of lower molecular weight was seen in some extracts.

Figure 4

FACS analysis of growth rate using fluorescent dye CFDA-SE: The first graph (green) in each panel shows fluorescent cells at 0 hour time point and the second (black) shows a decrease in fluorescence at 72 hr after the cells proliferate. There is no difference in the growth rate between the parental cell line and the transfected one.

Figure 5

UDG activity in mitochondrial extracts of parental MCF12A cells and transfected cell lines: Lanes 1, 3, 5 and 7 show UDG activity in mitochondrial extracts from MCF12A parental cell line, cells transfected with pCMV UNG, cells transfected with pCMV UGI, cells transfected with pCMV/myc/mito control vector and commercially available UDG enzyme, that acted as a positive control, respectively. Lanes 2, 4, 6, 8 and 10 shows an inhibition of UDG activity when commercially available Ugi was added in mitochondrial extracts from MCF12A parental cell line, cells transfected with pCMV UNG, cells transfected with pCMV UGI, cells transfected with pCMV/myc/mito control vector and commercially available UDG enzyme, that acted as a positive control, respectively.