Biology subjects

Forslund, J. M. E.

Publications and source records attributed to Forslund, J. M. E..

3 recordsLinked to original sources

bioRxiv2024

The Y951N patient mutation inactivates the intramolecular switch in human mitochondrial DNA POLgamma

Mitochondrial DNA (mtDNA) stability, essential for cellular energy production, relies on DNA polymerase gamma (POL{gamma}). Here, we show that the POL{gamma} Y951N disease causing mutation induces replication stalling and severe mtDNA depletion. However, unlike other POL{gamma} disease causing mutations, Y951N does not directly impair exonuclease activity and only mildly affects polymerase activity. Instead, we found that Y951N compromises the enzymes ability to efficiently toggle between DNA synthesis and degradation, and is thus the first patient-derived mutation with impaired polymerase-exonuclease switching. These findings provide new insights into the intramolecular switch when POL{gamma} proofreads the newly-synthesized DNA strand, and reveal a new mechanism for causing mitochondrial DNA instability. Significance StatementDNA polymerase gamma (POL{gamma}) is essential for copying mitochondrial DNA (mtDNA), which is crucial for our energy production. POL{gamma} must accurately switch between making new DNA (polymerase activity) and correcting errors (exonuclease activity). While it is known that mutations in POL{gamma} can cause mitochondrial diseases by directly impairing these enzymatic functions, this study reveals a new mechanism. The Y951N mutation disrupts POL{gamma}s ability to switch between these activities, leading to severe blockages in DNA replication and a loss of mtDNA in human cells, even without significant direct impairment of polymerase or exonuclease activities. These findings provide new insights into the origins of mitochondrial diseases.

biochemistry↗

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

The specific AMPK activator A-769662 ameliorates pathological phenotypes following mitochondrial DNA depletion

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis that also plays a role in preserving mitochondrial function and integrity. Upon a disturbance in the cellular energy state that increases AMP levels, AMPK activity promotes a switch from anabolic to catabolic metabolism to restore energy homeostasis. However, it is currently unclear how severe of a mitochondrial dysfunction is required to trigger AMPK activation, and whether stimulation of AMPK using specific agonists can improve the cellular phenotype following mitochondrial dysfunction. Using a cell model of mitochondrial disease characterized by progressive mitochondrial DNA (mtDNA) depletion and deteriorating mitochondrial metabolism, we show that mitochondria-associated AMPK becomes activated early in the course of the advancing mitochondrial dysfunction, before any quantifiable decrease in the ATP/(AMP+ADP) ratio or respiratory chain activity. Moreover, stimulation of AMPK activity using the specific small-molecule agonist A-769662 alleviated the mitochondrial phenotypes caused by the mtDNA depletion and restored normal mitochondrial membrane potential. Notably, the agonist treatment was able to partially restore mtDNA levels in cells with severe mtDNA depletion, while it had no impact on mtDNA levels of control cells. The beneficial impact of the agonist was also observed in cells from patients suffering from mtDNA depletion. However, the positive effects of A-769662 in the two experimental cell models appeared to involve at least partially different mechanisms. These findings improve our understanding of the effects of specific small-molecule activators of AMPK on mitochondrial and cellular function, and suggest a potential utility for these compounds in disease states involving mtDNA depletion.

molecular biology↗