Project description:Studying whether removal of base-excision repair from mitochondria will result into increase in mitochondrial DNA (mtDNA) mutation load. The endogenous genes of OGG1 and MUTYH DNA glycosylases were modified to lack the genomic region encoding for the predicted mitochondrial targeting sequence. The mouse lines used: A mouse line that lacks the region encoding for the mitochondrial targeting sequence (L2 to W23) of OGG1 (Ogg1 dMTS mice). A mouse line that lacks the region encoding the mitochondrial targeting sequence (K2 to P33) of MUTYH (Mutyh dMTS mice). To accumulate mutations to the mitochondrial DNA these mice were bred double homozygous Mutyh dMTS x Ogg1 dMTS mice as a maternal lineage for five consecutive generations and mitochondrial DNA from liver was extracted from the offspring and sequenced with Illumina. OGG1 and MUTYH are involved in repair of 8-oxo-dG from DNA. 8-oxo-dG can be a mutagenic lesion because some DNA repair polymerases are known to erroneously incorporate adenosine opposite to 8-oxo-dG during replication leading to GC>TA transversion mutations.
Project description:To study whether increase in mitochondrial oxidative stress (SOD2 removal) and decrease in mitochondrial DNA repair (Ogg1 dMTS) results into increase in mitochondrial DNA mutation load. Oxidative stress has been suggested to induce mutations in mtDNA. To verify this, we extracted and sequenced (Illumina) mitochondrial DNA from heart Sod2 knockout animals that were also deficient for mitochondrial base-excision repair. The repair deficiency was induced by removing the genomic region encoding for the predicted mitochondrial targeting sequence from endogenous OGG1 (L2 to W23) called Ogg1 dMTS mice, thus excluding the protein from mitochondria. OGG1 is a DNA glycosylase that recognizes and repairs 8-oxo-dG damage from DNA. Oxidative stress can induce 8-oxo-dG lesions, thus we removed the mitochondrial matrix localized superoxide dismutase (SOD2) from these mice to increase the level of oxidative stress. 8-oxo-dG lesion can be mutagenic because some DNA repair polymerases are known to erroneously incorporate adenosine opposite to 8-oxo-dG during replication leading to GC>TA transversion mutations.