The Saccharomyces cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro

Abstract

Telomere length homeostasis is vital for maintaining genomic stability and is regulated by multiple factors, including telomerase activity and DNA helicases. The Pif1 helicase was the first discovered catalytic inhibitor of telomerase, but recent experimental evidence suggests that Hrq1, the yeast homolog of the disease-linked human RecQ-like helicase 4 (RECQL4), plays a similar role via an undefined mechanism. Using yeast extracts enriched for telomerase activity and an primer extension assay, here we determined the effects of recombinant WT and inactive Hrq1 and Pif1 on total telomerase activity and telomerase processivity. We found that titrations of these helicases alone have equal-but-opposite biphasic effects on telomerase, with Hrq1 stimulating activity at high concentrations. When the helicases were combined in reactions, however, they synergistically inhibited or stimulated telomerase activity depending on which helicase was catalytically active. These results suggest that Hrq1 and Pif1 interact and that their concerted activities ensure proper telomere length homeostasis We propose a model in which Hrq1 and Pif1 cooperatively contribute to telomere length homeostasis in yeast.

The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain

“…We recently found that Pif1 impacts in vitro telomerase activity in a biphasic manner: low concentrations of Pif1 increase telomerase activity, but high Pif1 concentrations decrease telomerase activity [14]. Using a 15-nt telomerase repeat sequence (TG 1-3 ) oligonucleotide substrate (Tel15), an S. cerevisiae extract enriched for telomerase activity, and recombinant Pif1, we demonstrated a similar phenomenon here in Figure 2A,B.…”

“…Chemical reagents were purchased from Sigma or DOT Scientific (Burton, MI, USA). All recombinant proteins were purified as described in [14].…”

“…This plasmid was also used as the template to create pSUMO-Pif1ΔN to overexpress an N-terminal truncation of Pif1 lacking the first 233 amino acids of the helicase (Pif1ΔN) in E. coli. The overexpression and purification of these proteins proceeded as previously described for full-length Pif1 [14]. Recombinant Hrq1 was generated using baculovirus-infected insect cells as previously described [6,18].…”

“…In vitro telomerase assays were performed as described in [14] using telomerase-enriched extracts from S. cerevisiae prepared by DEAE fractionation of clarified lysates [19,20]. Each telomerase preparation was titrated to standardize activity levels before use in experiments.…”

Yeast Genome Maintenance by the Multifunctional PIF1 DNA Helicase Family

“…Just recently, Hrq1, a DNA helicase that has similarity to the human RecQ helicase RecQL4 [142], was shown to functionally interact with ScPif1 to synergistically modulate telomere length homeostasis by regulating telomerase activity [143]. Depending on whether the ScPif1 helicase or the Hrq1 helicase was active, telomerase was either inhibited or stimulated, and the authors propose that post-translational modifications that modulate the helicase activity of either one, such as already shown for ScPif1 [144,145], could thus regulate telomerase activity [143].…”