Baskerville, V. | Search bioRxiv

bioRxiv2023

G2C4 targeting antisense oligonucleotides potently mitigate TDP-43 dysfunction in C9orf72 ALS/FTD human neurons

The G4C2 repeat expansion in the C9orf72 gene is the most common genetic cause of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Many studies suggest that dipeptide repeat proteins produced from this repeat are toxic, yet, the contribution of repeat RNA toxicity is under investigated and even less is known regarding the pathogenicity of antisense repeat RNA. Recently, two clinical trials targeting G4C2 (sense) repeat RNA via antisense oligonucleotide failed despite a robust decrease in sense encoded dipeptide repeat proteins demonstrating target engagement. Here, in this brief report, we show that G2C4 antisense, but not G4C2 sense, repeat RNA is sufficient to induce TDP-43 dysfunction in induced pluripotent stem cell (iPSC) derived neurons (iPSNs). Unexpectedly, only G2C4, but not G4C2 sense strand targeting, ASOs mitigate deficits in TDP-43 function in authentic C9orf72 ALS/FTD patient iPSNs. Collectively, our data suggest that the G2C4 antisense repeat RNA may be an important therapeutic target and provide insights into a possible explanation for the recent G4C2 ASO clinical trial failure.

neuroscience↗

bioRxiv2022

Rapid, DNA-induced subunit exchange by DNA gyrase

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. GyrA is usually found as a dimer in solution, whereas GyrB can exist as a monomer. DNA gyrase is able to loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion of a positive loop into a negative one, thereby introducing negative supercoiling into the bacterial genome, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface "swapping" or "exchange" (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface exchange. This exchange does not require ATP, can occur in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping also explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed "swivelling" mechanism for DNA gyrase (Gubaev, Weidlich, and Klostermeier 2016).

biochemistry↗