Functional characterization of two variants of mitochondrial topoisomerase TOP1MT that impact regulation of the mitochondrial genome

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT

“…Live cell imaging was done on 35 mm glass bottom dishes, 8 × 10 4 cells were seeded and grown for two days. MitoTracker Red (50 nM) (Thermo Fisher Scientific, M7512) and PicoGreen (Thermo Fisher Scientific, P7581) (3 μL/mL) were used to visualize mitochondrial networks and mtDNA nucleoids respectively [39,53].…”

“…Given the mitochondrial dysfunction noted in patient-derived blood cells (Fig S2 ), we examined the ability of the TOP1MT P193L variant to rescue multiple mitochondrial parameters impaired in HCT116 KO cells [39]. As described previously [39], starting from HCT116 WT and HCT116 KO, we generated four different stable cell lines using retroviral constructs expressing TOP1MT and the mCherry fluorescent protein separated by a self-cleavage peptide (TOP1MT-T2A-mCherry), or an empty vector control. This construct leads to expression of TOP1MT, which will be directed to the mitochondria by the mitochondrial targeting sequence, and mCherry, which can be used to monitor transduction.…”

“…Next, we wanted to determine the ability of the P193L variant to rescue the aberrant functions previously identified in HCT116 KO cells [39], including: reduced nucleoid size, the hyperfused mitochondrial network, and decreases in mitochondrial transcription, mtDNA replication, mitochondrial translation and oxidative phosphorylation.…”

“…Based on our previous work [39], and the role of TOP1MT in preventing negative supercoiling of mtDNA [68] we examine mtDNA. To look at supercoiling, we used a southwestern blot approach that labels mtDNA with BrdU, which can then be separated on an agarose gel, transferred to a membrane and visualized by immunoblotting.…”

“…One intriguing candidate is TOP1MT, a topoisomerase that helps resolve crossovers and knots that can originate during maintenance of the circular mtDNA genome. TOP1MT is the only human topoisomerase that is solely present in the mitochondria [33][34][35], where it plays important roles in mtDNA replication, transcription, and translation [36][37][38][39]. Interestingly, a wide range of autoantibodies have been described and detected in the serum of SLE patients [40,41], including anti double stranded DNA (anti dsDNA) [42,43] and autoantibodies recognizing the nuclear topoisomerase enzyme TOP1 [44][45][46][47] as well as the mitochondrial TOP1MT [48].…”