Biology subjects

Bader, C. P. J.

Publications and source records attributed to Bader, C. P. J..

3 recordsLinked to original sources

bioRxiv2024

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.

biochemistry↗

bioRxiv2024

A small molecule inhibitor Mirin prevents TOP3A-dependent mtDNA breakage and segregation

Mirin, the chemical inhibitor of MRE11, has been recently reported to prevent immune response activation caused by mitochondrial DNA (mtDNA) breakage and release upon replication stalling. We show here that Mirin prevents mitochondrial replication fork breakage in mitochondrial 3-exonuclease MGME1 deficient cells and the resulting innate immune response induction, but that this occurs independently of MRE11. Furthermore, Mirin also caused alteration of mtDNA supercoiling and accumulation of hemicatenated replication termination intermediates, hallmarks of topoisomerase dysfunction, as well as alleviated topological changes induced by the overexpression of mitochondrial TOP3A, including TOP3A-dependent strand breakage at the non-coding region of mtDNA, potentially explaining its protective effect in the MGME1-knockout cells. Although Mirin does not inhibit TOP3A in vitro, our results demonstrate its MRE11-independent effects in cells and give insight into the mechanisms of mtDNA segregation, as well as the maintenance of genomic integrity in mitochondria. Significance StatementO_LIBroken mitochondrial DNA (mtDNA) in MGME1 knockout cells activates innate immune response, which is prevented by Mirin, a small molecule inhibitor of MRE11. C_LIO_LIMirin also interferes with mtDNA replication termination and segregation, suggesting that termination intermediates or paused forks are a major source of mtDNA breakage. C_LIO_LIWe show that these effects are likely dependent on topoisomerase 3A (TOP3A) -related processes in mitochondria, questioning the Mirin target also in the nucleus. C_LI

molecular biology↗

bioRxiv2024

PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded Type 4 Secretion Systems (T4SS). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many their function remains unclear. Here, we characterize PrgE, a proposed SSB from Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very uncharacteristic DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string, and this plasticity of PrgEs oligomerization is further confirmed by in vitro studies. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from the eukaryotic SSBs.

biochemistry↗