Protein RPA - Telomere Maintenance

Human RPA Emerges as Key Factor in Telomere Maintenance

New research suggests that replication protein A (RPA), a protein complex long known for its roles in DNA replication and repair, also serves as an essential stimulator of telomerase activity. Results indicate that RPA enhances the enzyme's ability to add repeated DNA sequences to chromosome ends, a process critical for preventing telomere shortening. This discovery, detailed in a study from the University of Wisconsin-Madison, provides insights into mechanisms that support telomere maintenance and offers potential avenues for promoting healthier aging.

The Fundamentals of Telomeres and Telomerase

Telomeres are protective caps at the ends of linear chromosomes, consisting of repetitive DNA sequences that safeguard genetic material from degradation. As cells divide, these caps naturally shorten, eventually triggering cellular aging if not maintained. Telomerase—the enzyme responsible for extending telomeres—counteracts this erosion by adding DNA repeats to the G-rich single-stranded overhangs.

Shelterin proteins, such as TPP1 and POT1, have been recognized for recruiting telomerase to telomeres and stimulating its repeat addition processivity (RAP), the measure of how many repeats the enzyme can add in one binding event. However, the in vivo significance of this stimulation remained unclear until recent investigations into RPA.

RPA's Role in Enhancing Telomerase Processivity

Using in vitro primer-extension assays, researchers found that human RPA increases telomerase RAP by approximately six-fold, an effect comparable to that of the TPP1-POT1 complex but achieved through distinct mechanisms. Results indicated that this stimulation requires the full RPA heterotrimer and its single-stranded DNA (ssDNA)-binding domains, without altering overall telomerase activity.

Further experiments with competitors like polyT oligonucleotides confirmed RPA's dependence on ssDNA binding. AlphaFold modeling predicted a direct interaction between RPA's OB-D domain and telomerase's TERT subunit, a finding supported by non-linear processivity enhancements at sub-stoichiometric RPA concentrations and co-immunoprecipitation assays showing RPA-TERT associations in cells.

Separating Functions Through Targeted Mutations

To distinguish RPA's binding site from TPP1's, the team created separation-of-function (SOF) TERT mutants guided by molecular dynamics simulations. These mutants selectively abolished RPA-mediated stimulation while preserving TPP1-POT1 effects, and vice versa. Immunofluorescence revealed that RPA localizes to telomeres via telomerase without activating DNA damage responses, as evidenced by the absence of RPA32 phosphorylation or 53BP1 foci.

In cellular models, SOF mutants defective in RPA stimulation led to telomere shortening, even when recruitment remained intact. This underscores RPA's indispensable contribution to telomere elongation.

Implications for Healthy Aging and Longevity

The study highlights RPA's role in supporting telomerase processivity, which helps maintain telomere length—a factor associated with extended lifespan and healthier aging. Results suggest that longer telomeres correlate with delayed cellular senescence, reduced oxidative stress, and potentially increased life expectancy in humans.

For instance, research indicates that healthy lifestyles, including strength training, are linked to longer telomeres and improved biological aging markers. Interventions aimed at preserving or enhancing telomere maintenance, such as optimizing RPA-telomerase interactions, could delay aging processes and promote longevity. One analysis proposes that increasing telomere length in individuals with shorter ones might extend lifespan by several years.

RPA-like complexes in other species also modulate telomerase, suggesting an evolutionarily conserved mechanism that underpins robust telomere function across life forms. "RPA’s function in helping maintain long, healthy telomeres in humans has been confirmed through experimental validation," notes the research team.

Future Implications

These findings offer encouraging insights into telomere biology, potentially guiding strategies for enhanced healthy aging. However, further studies are needed to explore coordination with other telomere factors. As Lim mentions, this work fosters a collaborative path toward better understanding molecular processes that support vitality.

"This line of research goes beyond a biochemical understanding of a molecular process.  It deepens clinical understanding of telomere diseases" - Ci Ji Lim