RPA Joins the Telomerase Team: New Complex Revealed in Fission Yeast

Every time a human cell divides, the ends of its chromosomes—a protective stretch of repetitive DNA called the telomere—grow a little shorter. Left unchecked, this erosion would eventually strip chromosomes of the caps they need to survive, which is why the enzyme telomerase exists. Telomerase extends telomeres, counteracting the inevitable attrition of replication, but the enzyme cannot simply wander up to a chromosome end and start working. It must first be recruited to the telomere, and then, crucially, it must be switched into a productive state that actually lengthens the DNA. That second step—how a recruited telomerase becomes an active one—has been one of the most stubborn mysteries in chromosome biology, and a new study in PLOS Genetics offers the most detailed answer yet.

A team led by Bettina Moser and Toru Nakamura, working with colleagues including Madeline Points, Sourav Agrawal, Adam Didier, Amanda Mennie, Ci Ji Lim and Yong-jie Xu, has assembled genetic and structural evidence for a three-part molecular machine in fission yeast: a complex containing the single-stranded DNA-binding protein RPA, the telomerase reverse transcriptase Trt1 (the yeast equivalent of human TERT), and Tpz1, the yeast counterpart of the mammalian telomere protein TPP1. Their findings, published under the title describing the RPA–TERT–Tpz1 complex as a promoter of telomere extension and a suppressor of telomere recombination, suggest that RPA is not merely a bystander at chromosome ends but an essential structural component of the active telomerase holoenzyme.

The significance of this work lies in how it reframes the telomerase activation problem. For years, researchers focused on the recruitment step: telomerase is delivered to telomeres through interactions between TERT and TPP1 in mammals, or between the telomerase subunit Est3 and the TPP1-like protein in budding yeast. But recruitment alone does not guarantee that telomerase will add DNA repeats. Recent studies in humans and budding yeast had hinted that Replication Protein A, better known for its role in DNA replication and repair, also stimulates telomerase by physically contacting TERT in human cells and the TPP1 ortholog in budding yeast. The new study takes that hint and turns it into a comprehensive molecular model.

The researchers began with a genetic screen in fission yeast, a single-celled organism that has long served as a powerful model for telomere biology because its telomere maintenance machinery closely mirrors that of humans. Mutations that disrupted telomere maintenance surfaced in the screen, and rather than stopping at the list of affected genes, the team turned to AlphaFold3, the artificial intelligence system that predicts protein structures and their interactions, to model how RPA, Trt1 and Tpz1 might fit together. The computational models then guided systematic mutagenesis: the researchers altered specific amino acids in each of the three proteins and asked how the changes affected telomere length and chromosome stability in living yeast cells.

The combined approach revealed four distinct protein-protein interfaces that together support telomerase function. Two of them connect RPA to Trt1: one involving the Ssb1 subunit of RPA (the equivalent of the human RPA1 subunit) and one involving the Ssb2 subunit (equivalent to human RPA2). A third interface links the Ssb2 subunit of RPA directly to Tpz1. The fourth is the previously characterized interaction between Trt1 and Tpz1, mediated by the so-called TEL patch—a cluster of acidic amino acids on TPP1-like proteins that is known to dock TERT. In other words, the active telomerase complex appears to be held together by a web of contacts in which RPA physically bridges the telomere-binding protein Tpz1 and the enzyme Trt1.

One of the most striking results concerns a specific amino acid in Tpz1, arginine 81. Earlier work had assigned this residue to the TEL patch, implying that it contacted Trt1 directly. The new structural modeling and mutagenesis data tell a different story: Tpz1-R81 instead reaches across to contact Ssb2, the RPA subunit. This reassignment matters because it changes the wiring diagram of the telomerase activation machine. Rather than a simple two-component handshake between the telomere anchor and the enzyme, the picture is now a ternary complex in which RPA occupies a central coordinating position, touching both partners simultaneously.

Genetic evidence backs up the structural model. Using epistasis analysis—a technique that tests whether mutations in different genes affect the same biological pathway—and suppressor analysis, in which a second mutation can be engineered to compensate for the first, the researchers showed that the newly identified RPA-Trt1 and RPA-Tpz1 interfaces collaborate with the Trt1-Tpz1 interface to enable telomerase activation after the enzyme has already been recruited to the telomere. Disrupting any one of these contacts compromises telomere extension, and the pattern of genetic interactions indicates that the interfaces function cooperatively rather than redundantly. The complex, in short, behaves like a multi-point scaffold that must be assembled correctly before telomerase can do productive work.

The study also uncovered a second, equally important function: keeping telomeres honest. When telomerase cannot extend chromosome ends, cells sometimes resort to recombination-based pathways that copy telomeric DNA from one chromosome to another. These recombination-based survival mechanisms are genomically destabilizing and, in human cells, are associated with the alternative lengthening of telomeres seen in many cancers. The fission yeast experiments showed that the RPA-Trt1-Tpz1 complex suppresses such recombination while promoting legitimate telomerase-mediated extension. The same molecular machine, therefore, performs a dual quality-control role: it turns on the proper lengthening enzyme and shuts down the error-prone backup pathway.

Perhaps the most consequential finding is evolutionary. Comparative AlphaFold3 analyses suggest that the interaction network identified in fission yeast is likely conserved in budding yeast and in humans. If the RPA-TERT-TPP1 ternary architecture holds across such distant branches of life, then RPA’s role as a core activator of telomerase is probably not a yeast quirk but a fundamental feature of eukaryotic chromosome-end maintenance. That has immediate implications for human biology: telomerase activation is a hallmark of roughly ninety percent of cancers, and drugs targeting the enzyme’s recruitment interface are under active investigation. If RPA contacts are required for productive telomerase engagement in human cells, those contacts represent an additional layer of the enzyme’s control system—and potentially an additional drug target.

The work also resolves a conceptual tension in the field. RPA binds single-stranded DNA and is abundant at telomeres, where the G-rich overhang provides an obvious binding substrate. Earlier models treated RPA’s presence at chromosome ends as incidental, or as a competitor that must be displaced for telomerase to act. The new data support the opposite view: RPA is an active participant, physically coordinating the telomere anchor and the enzyme so that recruitment is converted into extension. In this view, the telomerase holoenzyme at a chromosome end is a larger and more intricately assembled machine than the classic textbook depiction of TERT, its RNA template and a telomere anchor alone. As with many recent discoveries powered by AI-guided structural prediction combined with classical genetics, the lesson is that molecular machines often hide their most important players until someone looks at the whole assembly at once. With RPA now firmly installed at the heart of the active telomerase complex, the next challenge is to determine exactly how these interfaces are regulated through the cell cycle, and whether human cancers depend on the same contacts to keep their chromosomes endlessly young.

Subject of Research: Structure and function of the RPA–TERT–Tpz1 telomerase activation complex in fission yeast telomere maintenance

Article Title: Fission yeast RPA–TERT–Tpz1 TPP1 complex promotes telomere extension and suppresses telomere recombination

Article References: Fission yeast RPA–TERT–Tpz1 TPP1 complex promotes telomere extension and suppresses telomere recombination. (n.d.). https://doi.org/10.1371/journal.pgen.1012297

Image Credits: AI Generated

DOI: 10.1371/journal.pgen.1012297

Keywords: telomerase, telomeres, RPA, TERT, Tpz1, TPP1, fission yeast, telomere extension, telomere recombination, AlphaFold3, chromosome biology, PLOS Genetics