Biochemical analysis of the endoribonuclease activity of the human mitochondrial topoisomerase 1

The incorporation of ribonucleotides (rNMPs) into the nuclear genome leads to severe genomic instability, including strand breaks and short 2-5 bp deletions at repetitive sequences. Curiously, the detrimental effects of rNMPs are not observed for the human mitochondrial genome (mtDNA) that typically contains several rNMPs per molecule. Given that the nuclear genome instability phenotype is dependent on the activity of the nuclear topoisomerase 1 enzyme (hTop1), and mammalian mitochondria contain a distinct topoisomerase 1 paralog (hTop1mt), we hypothesized that the differential effects of rNMPs on the two genomes may reflect differing properties of the two cellular topoisomerase 1 enzymes. Here, we characterized the endoribonuclease activity of hTop1mt and found it to be less efficient than that of its nuclear counterpart, a finding that was partly explained by its substrate binding properties. While hTop1 and yeast Top1 showed higher affinity for an rNMP-containing substrate and were able to cleave at an rNMP located outside of the consensus cleavage site, hTop1mt showed no preference for rNMPs. As a consequence, hTop1mt was inefficient at producing the short rNMP-dependent deletions that are characteristic of Top1-driven genome instability. These findings help explain the tolerance of rNMPs in the mitochondrial genome.