Xu, Y.-j. | bioRxiv Science

bioRxiv2026

Fission yeast RPA-TERT-Tpz1TPP1 complex promotes telomere extension and suppresses telomere recombination

Telomerase maintains chromosome ends by extending telomeric DNA, yet how recruited telomerase becomes productively engaged remains poorly understood. Recent studies found that Replication Protein A (RPA) contributes to telomerase stimulation through interaction with TERT in humans and with the TPP1 ortholog Est3 in budding yeast, suggesting a direct role in telomerase activation. Here, we provide genetic and structural modeling evidence for a RPA-Trt1TERT-Tpz1TPP1 ternary complex that promotes telomere extension while suppressing recombination in fission yeast. Guided by results from genetic screen, followed by AlphaFold3 modeling and systematic mutagenesis of RPA, Trt1, and Tpz1, we identify four key interfaces supporting telomerase function: Ssb1RPA1-Trt1, Ssb2RPA2-Trt1, Ssb2RPA2-Tpz1, and the TEL-patch-mediated Trt1-Tpz1 interaction. Notably, Tpz1-R81, previously assigned as the TEL patch, instead contacts Ssb2 in the complex. Epistasis and suppressor analyses indicate that the newly identified RPA-Trt1 and RPA-Tpz1 interfaces collaborate with the Trt1-Tpz1 interface to allow telomerase activation after recruitment. Furthermore, comparative analyses using AlphaFold3 suggest that these interactions are likely conserved in budding yeast and humans. Collectively, these findings support a model in which RPA serves as an essential component of the active telomerase complex, coordinating TERT and TPP1-like factors to enable productive telomerase engagement. Author SummaryTelomeres are specialized dynamic protective structures at the ends of eukaryotic chromosomes that must be properly maintained to preserve genome stability. Telomerase extends telomeric DNA, but how recruited telomerase becomes fully activated to promote telomere extension remains poorly understood. In this study, we use fission yeast to investigate the role of the conserved single-stranded DNA-binding protein complex Replication Protein A (RPA) in this process. We find that RPA forms a functional complex with the telomerase catalytic subunit TERT and Tpz1, a component of the telomere protection complex shelterin and the fission yeast ortholog of human TPP1. Genetic and structural analyses identify multiple interactions within the RPA-TERT-Tpz1 complex that are required for efficient telomere extension. Disrupting these interactions allows telomerase recruitment but prevents productive telomerase action at chromosome ends. Our results further suggest that similar mechanisms may operate in other organisms, including budding yeast and humans. These findings provide insight into how telomerase activity is regulated at chromosome ends.

molecular biology↗

bioRxiv2020

The RecQ DNA helicase Rqh1 promotes Rad3ATR kinase signaling in the DNA replication checkpoint pathway of fission yeast

Rad3 is the orthologue of ATR and the sensor kinase of the DNA replication checkpoint in Schizosaccharomyces pombe. Under replication stress, it initiates checkpoint signaling at the forks necessary for maintaining genome stability and cell survival. To better understand the checkpoint initiation process, we have carried out a genetic screen in fission yeast by random mutation of the genome looking for mutants with defects in Rad3 kinase signaling. In addition to the previously reported tel2-C307Y mutant (1), this screen has identified six mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 mutations except a start codon mutation are all in the helicase domain, indicating that the helicase activity of Rqh1 plays an important role in the replication checkpoint. In support of this notion, integration of two helicase-inactive mutations or deletion of rqh1 generated a similar Rad3 signaling defect and heterologous expression of human RECQ1, BLM and RECQ4 restored the Rad3 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication checkpoint function of Rqh1 is highly conserved and mutations in the helicase domain of these human enzymes may cause the checkpoint defect and contribute to the cancer predisposition syndromes.

cell biology↗