Biology subjects

Nageshan, R. K.

Publications and source records attributed to Nageshan, R. K..

2 recordsLinked to original sources

bioRxiv2023

Parallel genetic screens identify nuclear envelope homeostasis as a key determinant of telomere entanglement resolution in fission yeast

In fission yeast lacking the telomere binding protein, Taz1, replication stalls at telomeres, triggering deleterious downstream events. Strand invasion from one taz1{Delta} telomeric stalled fork to another on a separate (non-sister) chromosome leads to telomere entanglements, which are resolved in mitosis at 32{degrees}C; however, entanglement resolution fails at [≤]20{degrees}C, leading to cold-specific cell lethality. Previously, we found that loss of the mitotic function of Rif1, a conserved DNA replication and repair factor, suppresses cold sensitivity by promoting resolution of entanglements without affecting entanglement formation. To understand the underlying pathways of mitotic entanglement resolution, we performed a series of genomewide synthetic genetic array screens to generate a comprehensive list of genetic interactors of taz1{Delta} and rif1{Delta}. We modified a previously described screening method to ensure that the queried cells were kept in log phase growth. In addition to recapitulating previously identified genetic interactions, we find that loss of genes encoding components of nuclear pore complexes (NPCs) promotes telomere disentanglement and suppresses taz1{Delta} cold sensitivity; we attribute this to more rapid anaphase midregion nuclear envelope (NE) breakdown in the absence of these NPC components. Moreover, loss of genes involved in lipid metabolism reverses the ability of rif1+ deletion to suppress taz1{Delta} cold sensitivity, again pinpointing NE modulation. A rif1+ separation-of-function mutant that specifically loses Rif1s mitotic functions yields similar genetic interactions. Genes promoting membrane fluidity were enriched in a parallel taz1+ synthetic lethal screen at permissive temperature, cementing the idea that the cold specificity of taz1{Delta} lethality stems from altered NE homeostasis.

genetics↗

bioRxiv2023

Fate of telomere entanglements is dictated by the timing of anaphase midregion nuclear envelope breakdown

Persisting replication intermediates can confer mitotic catastrophe if left unresolved. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork stalling and in turn, telomere entanglements that stretch between the segregating chromosomes at anaphase. At [≤]20{degrees}C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, localizes between the segregating chromosomes and specifically hinders the resolution of telomere entanglements without affecting their formation. During anaphase, the spindle and the last segments of segregating chromatin are encased by the nuclear envelope (NE), creating a microdomain termed the anaphase midregion. In the final stages of fission yeast mitosis, this midregion undergoes local NE breakdown. Here we demonstrate that in response to taz1{Delta} telomeric entanglements, Rif1 delays midregion NE breakdown, and this delay disfavors entanglement resolution. Accordingly, gene deletions that hasten midregion NE breakdown phenocopy, and are epistatic with, rif1+ deletion. Conversely, gene deletions that delay midregion NE breakdown block the taz1{Delta} telomere detanglement afforded by loss of Rif1. Overexpression of Rif1 in a wild type background causes cold-specific NE defects and lethality, which are rescued by treatment with a membrane fluidizing agent. We propose that delayed NE breakdown normally favors the resolution of simple entanglements, arising from incomplete replication, by delaying exposure to the cytoplasm. In contrast, resolution of more complex entanglements involving strand invasion, like entanglements between nonsister taz1{Delta} telomeres, requires more rapid exposure to the cytoplasm. These observations uncover an unexpected coordination between NE remodeling and DNA processing events that can prevent or promote aneuploidy.

molecular biology↗