Rnr1, but not Rnr3, facilitates the sustained telomerase-dependent elongation of telomeres

Abstract

Ribonucleotide reductase (RNR) provides the precursors for the generation of dNTPs, which are required for DNA synthesis and repair. Here, we investigated the function of the major RNR subunits Rnr1 and Rnr3 in telomere elongation in budding yeast. We show that Rnr1 is essential for the sustained elongation of short telomeres by telomerase. In the absence of Rnr1, cells harbor very short, but functional, telomeres, which cannot become elongated by increased telomerase activity or by tethering of telomerase to telomeres. Furthermore, we demonstrate that Rnr1 function is critical to prevent an early onset of replicative senescence and premature survivor formation in telomerase-negative cells but dispensable for telomere elongation by Homology-Directed-Repair. Our results suggest that telomerase has a "basal activity" mode that is sufficient to compensate for the “end-replication-problem” and does not require the presence of Rnr1 and a different "sustained activity" mode necessary for the elongation of short telomeres, which requires an upregulation of dNTP levels and dGTP ratios specifically through Rnr1 function. By analyzing telomere length and dNTP levels in different mutants showing changes in RNR complex composition and activity we provide evidence that the Mec1ATR checkpoint protein promotes telomere elongation by increasing both dNTP levels and dGTP ratios through Rnr1 upregulation in a mechanism that cannot be replaced by its homolog Rnr3.

A genetic screen pinpoints ribonucleotide reductase residues that sustain dNTP homeostasis and specifies a highly mutagenic type of dNTP imbalance

“…Interestingly, supporting the idea that dGTP levels may play a central role for genome stability, previous work (56,57) reported a positive correlation between intracellular dGTP levels and both telomere length and telomerase processivity in vivo . Therefore, it seems that cells have evolved a way to take advantage of the least abundant dNTP (dGTP) to control key processes associated with genome maintenance.…”

Tel1 Activation by the MRX Complex Is Sufficient for Telomere Length Regulation but Not for the DNA Damage Response in Saccharomyces cerevisiae

“…MEC1 is an essential gene as mec1D cells are not able to activate dNTP production (Zhao et al 1998). mec1D cells survive only with codeletion of either SML1 or CRT1 (also called RFX1), and in mec1D sml1D or mec1D crt1D cells, dNTP production is increased (Gupta et al 2013;Maicher et al 2017). mec1D sml1D have telomeres similar to wild type, while mec1D crt1D have slightly shorter telomeres.…”

“…mec1D sml1D have telomeres similar to wild type, while mec1D crt1D have slightly shorter telomeres. This difference has been attributed to the fact that SML1 and CRT1 regulate different pathways of dNTP production (Maicher et al 2017). mec1-21 and mec1-14, Mec1 hypomorphic alleles, have telomeres slightly shorter than wild type (Ritchie et al 1999;Longhese et al 2000).…”

Histone depletion prevents telomere fusions in pre-senescent cells

“…Mec1 is essential to maintain the levels of dNTPs during replication and DNA damage, but can be eliminated without affecting viability in cells lacking Sml1, a Mec1-regulated inhibitor of the dNTPs synthesis [ 32 ]. Since the intracellular pool of dNTPs has a direct impact on telomere biology [ 33 – 35 ], all analyses were performed in sml1Δ strains except for tlc1Δ , which was compared with its isogenic wild-type strain. Wild-type and mec1Δ tel1Δ cells from streak 1 displayed similar levels of histone H4, which dropped two-fold in mec1Δ tel1Δ t :: HHF2 cells ( Fig 1A ).…”