Characterization of the telomerase modulating activities of yeast DNA helicases

Abstract

Eukaryotes with linear chromosomes circumvent the end replication problem via the action of a specialized ribonucleoprotein reverse transcriptase known as telomerase. Cells lacking telomerase activity will senesce when their chromosome ends shorten to a critical length. In contrast, cancer cells can become immortalized by upregulating telomerase to lengthen telomeres during each cycle of DNA replication. Thus, the regulation of telomerase is critical for normal telomere homeostasis. Of the various known ways that telomerase activity is modulated in vivo, recent studies have demonstrated that DNA helicases are involved. In Saccharomyces cerevisiae, the Hrq1 and Pif1 helicases act in a pathway that regulates telomerase extension at telomeres and at DNA double-strand DNA breaks. In vitro analysis demonstrates that when these helicases are combined in reactions, they synergistically inhibit or stimulate telomerase activity depending on which helicase is catalytically active. Here, we describe the methods for the overproduction and purification of Hrq1 and Pif1. We also report the preparation of partially purified cell extracts with telomerase activity and how the effects of these helicase on telomerase activity can be assessed in vitro.

Keywords: DNA helicase; Hrq1; PIF1; RecQ; Telomerase.

Fig. 1

The effects of Hrq1 and/or Pif1 on in vitro telomerase activity. (A) The wild-type helicases alone display differing effects on telomerase, but in combination, they inhibit telomerase primer extension significantly better than Pif1 alone. (B) The inactive Hrq1-K318A mutant has no effect on telomerase activity alone but, together with wild-type Pif1, is a potent telomerase inhibitor. (C) The inactive Pif1-K264A mutant has no effect on telomerase activity alone but, in the presence of wild-type Hrq1, greatly stimulates telomerase activity. *P <0.01, **P <0.001, and ***P <0.0001. This figure was adapted from Nickens, D. G., Rogers, C. M., & Bochman, M. L. (2018). The Saccharomyces cerevisiae Hrq1 and Pif1 DNA helicases synergistically modulate telomerase activity in vitro. The Journal of Biological Chemistry, 293(37), 14481–14496. doi:10.1074/jbc.RA118.004092.

Fig. 2

Generation of recombinant Hrq1. Hrq1 (~125kDa) was over-expressed in E. coli (A) or insect cells (B), and cell lysates were subjected to one step of purification over a Streptactin Sepharose column or in batch mode in the presence of Ni⁺² affinity resin, respectively. In both images, Lane 1 contains a sample of clarified cell lysate, and the following lanes contain 10-μL samples of elution fractions. Despite coming from 10-fold less cell mass, the insect cells yielded far more Hrq1 protein than E. coli.

Fig. 3

Titrating a fresh telomerase preparation. The ability of increasing amounts of a telomerase extract to perform primer extension of the Tel15 substrate is tested for each batch of extract prepared to standardize activity levels with previous experiments. The +1 to +7 nucleotide additions are noted in red, and we often see a prominent +5 extension. The markers are ³²P-labeled Tel15, Tel30, and Tel50 oligonucleotides. The addition of two amounts of BSA to the primer extension assay was also tested in this experiment to control for the effects of added protein to the reaction.