Human RPA is an essential telomerase processivity factor for maintaining telomeres.

Telomerase counteracts telomere shortening by repeatedly adding DNA repeats to chromosome ends. We identified the replication protein A (RPA) heterotrimer as a telomerase processivity factor critical for telomere maintenance. RPA stimulates telomerase processivity in vitro, and AlphaFold modeling predicts that RPA engages a telomerase surface distinct from the one bound by the shelterin subunit TPP1. Guided by these predictions, we engineered separation-of-function telomerase reverse transcriptase (TERT) mutants and found that the loss of RPA-mediated stimulation impairs telomere elongation, even when TPP1–POT1–mediated stimulation remains intact. Furthermore, short-telomere disease–associated TERT mutations reduce RPA-dependent telomerase stimulation, revealing a mechanistic link between impaired processivity and telomeropathies. Together, our findings establish human RPA as a key regulator of telomerase and offer molecular insights into telomere-related disease mechanisms. Editor's summary: Human replication protein A (RPA), long recognized as an essential factor in DNA replication and repair, has now been found to play a critical role at chromosome ends. Agrawal et al. demonstrated that RPA directly enhances the processivity of telomerase, the enzyme responsible for extending telomeres. By boosting telomerase's ability to add repeated DNA sequences, RPA proves vital for maintaining telomere length and genomic stability. This repurposing of a fundamental single-stranded DNA-binding complex provides fresh insights into telomere regulation and has implications for aging, cancer, and telomere-related disorders. —Di Jiang [ABSTRACT FROM AUTHOR]