Bader Cyrielle P J, Miyazaki-Kasho Erika, Forslund Josefin M E, Dash Aiswarya, Wessels Malgorzata, Wanrooij Paulina H
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden.
Nucleic Acids Res. 2025 Jun 6;53(11). doi: 10.1093/nar/gkaf475.
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 divergent 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 weaker affinity for its DNA substrate. Moreover, while hTOP1 and yeast TOP1 were able to cleave at an rNMP located even outside of the consensus cleavage site, hTOP1MT showed no such 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.
核糖核苷酸（rNMPs）掺入核基因组会导致严重的基因组不稳定，包括链断裂以及重复序列处2 - 5个碱基对的短缺失。奇怪的是，对于通常每个分子含有多个rNMPs的人类线粒体基因组（mtDNA），并未观察到rNMPs的有害影响。鉴于核基因组不稳定表型依赖于核拓扑异构酶1（hTOP1）的活性，而哺乳动物线粒体含有一种独特的拓扑异构酶1旁系同源物（hTOP1MT），我们推测rNMPs对这两个基因组的不同影响可能反映了两种细胞拓扑异构酶1的不同特性。在此，我们对hTOP1MT的核糖核酸内切酶活性进行了表征，发现其效率低于核对应物，这一发现部分归因于它对DNA底物的亲和力较弱。此外，虽然hTOP1和酵母TOP1能够在甚至位于共有切割位点之外的rNMP处切割，但hTOP1MT对rNMPs没有这种偏好。因此，hTOP1MT在产生由TOP1驱动的基因组不稳定所特有的短rNMP依赖性缺失方面效率低下。这些发现有助于解释线粒体基因组对rNMPs的耐受性。
