Telomere Length Homeostasis Responds to Changes in Intracellular dNTP Pools

Abstract

Telomeres, the ends of linear eukaryotic chromosomes, shorten due to incomplete DNA replication and nucleolytic degradation. Cells counteract this shortening by employing a specialized reverse transcriptase called telomerase, which uses deoxyribonucleoside triphosphates (dNTPs) to extend telomeres. Intracellular dNTP levels are tightly regulated, and perturbation of these levels is known to affect DNA synthesis. We examined whether altering the levels of the dNTP pools or changing the relative ratios of the four dNTPs in Saccharomyces cerevisiae would affect the length of the telomeres. Lowering dNTP levels leads to a modest shortening of telomeres, while increasing dNTP pools has no significant effect on telomere length. Strikingly, altering the ratio of the four dNTPs dramatically affects telomere length homeostasis, both positively and negatively. Specifically, we find that intracellular deoxyguanosine triphosphate (dGTP) levels positively correlate with both telomere length and telomerase nucleotide addition processivity in vivo. Our findings are consistent with in vitro data showing dGTP-dependent stimulation of telomerase activity in multiple organisms and suggest that telomerase activity is modulated in vivo by dGTP levels.A LL eukaryotes, as well as some prokaryotes with linear chromosomes, contain repetitive sequences called telomeres at the ends of their DNA. Telomeric DNA is bound by proteins that protect chromosome ends from being recognized as genotoxic DNA double-strand breaks in need of repair (Jain and Cooper 2010). However, telomeres shorten due to incomplete DNA replication and nucleolytic degradation. Left unchecked, this telomere erosion eventually results in very short, unprotected telomeres, leading to cell-cycle arrest and replicative senescence (Lundblad and Szostak 1989;Harley et al. 1990;Yu et al. 1990).Telomere shortening is counteracted by a specialized reverse transcriptase called telomerase (Greider and Blackburn 1985), whose core consists of a protein catalytic subunit and an RNA moiety-hTERT and hTR, respectively, in humans (Feng et al. 1995;Nakamura et al. 1997), and Est2 and TLC1, respectively, in the budding yeast Saccharomyces cerevisiae (Singer and Gottschling 1994;Lingner et al. 1997). Telomerase extends telomeres by repeated reverse transcription of a short sequence to the 39 ends of telomeres, using the RNA subunit as a template (Greider and Blackburn 1989;Yu et al. 1990;Singer and Gottschling 1994). Although the sequence of the telomeric repeats differs between species, a common feature is that they are all G-rich. In vertebrates, the repeat sequence is TTAGGG (Meyne et al. 1989), while in S. cerevisiae, the telomeric repeats have a consensus sequence of (TG) 0-6 TGGGTGTG(G) 0-1 (Forstemann and Lingner 2001).Deoxyribonucleoside triphosphates (dNTPs) are the building blocks of DNA, and their production needs to be tightly regulated as imbalances in dNTP pools can be mutagenic (Reichard 1988). In S. cerevisiae, the sole mode of dNTP production is through de novo dN...

Colon cancer-associated mutator DNA polymerase δ variant causes expansion of dNTP pools increasing its own infidelity

“…Deletion of CRT1 further increases dNTP pools in sml1Δ strains (58). We found that deletion of SML1 in dun1Δ pol3-R696W mutants restored dNTP pools to the levels observed in pol3-R696W strains (Fig.…”

“…Although dun1Δ mutants are not able to expand dNTP pools in response to the pol3-R696W mutation (Fig. 3B), the dNTP levels can be increased in dun1Δ strains by deleting the SML1 gene (58). RNR activity is also modulated by Crt1, a transcriptional repressor that binds to and inhibits transcription from RNR2, RNR3, and RNR4 promoters The data are based on DNA sequence analysis of 189 independent Can R mutants of msh6Δ pol3-R696W strains containing pPOL3.…”

The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells

“…The mechanism underlying the phenotype of the Acardi-Goutières syndrome caused by SAMHD1 mutations is still unknown, but the occasional occurrence of mitochondrial DNA deletions might be linked to dNTP pool abnormalities. The recently reported specific influence of the dGTP pool on telomere length homeostasis (38) further emphasizes the importance of keeping dGTP under control. Recent studies have shown that methylation of the SAMHD1 promoter regulates gene expression in human T cells (39) and that the protein is phosphorylated by the cyclin A2/CDK1 complex in cycling cells (40,41).…”

Novel Connections Between DNA Replication, Telomere Homeostasis, and the DNA Damage Response Revealed by a Genome-Wide Screen for TEL1/ATM Interactions in Saccharomyces cerevisiae

“…As telomere shortening has recently been shown to occur upon dNTP depletion (Gupta et al 2013), it is likely that the short telomeres in CCR4-NOT and 9-1-1 mutants are at least partially due to this mechanism. Consistent with a model for increased replication stress in tel1-D xxx-D cells, depleting nucleotide pools by pretreatment with HU (effectively phenocopying the loss of 9-1-1 or CCR4/ POP2) sensitizes tel1-D cells to MMS in a dose-dependent manner, whereas a wild-type strain is unaffected by the HU pretreatment (Figure 8).…”