The Ctf18RFC Clamp Loader Is Essential for Telomere Stability in Telomerase-Negative and mre11 Mutant Alleles

Abstract

The function of the replication clamp loaders in the semi-conservative telomere replication and their relationship to telomerase- and recombination mechanisms of telomere addition remains ambiguous. We have investigated the variant clamp loader Ctf18 RFC (Replication Factor C). To understand the role of Ctf18 at the telomere, we first investigated genetic interactions after loss of Ctf18 and TLC1 (the yeast telomerase RNA). We find that the tlc1▵ ctf18▵ double mutant confers a rapid >1000-fold decrease in viability. The rate of loss was similar to the kinetics of cell death in rad52▵ tlc1▵ cells. However, the Ctf18 pathway is distinct from Rad52, required for the repair of DSBs, as demonstrated by the synthetic lethality of rad52▵ tlc1▵ ctf18▵ triple mutants. These data suggest that each mutant elicits non-redundant defects acting on the same substrate. Second, interactions of the yeast hyper-recombinational mutant, mre11A470T, with ctf18▵ confer a synergistic cold sensitivity. The phenotype of these double mutants ultimately results in telomere loss and the generation of recombinational survivors. We observed a similar synergism between single mutants that led to hypersensitivity to the DNA alkylating agent, methane methyl sulphonate (MMS), the replication fork inhibitor hydroxyurea (HU), and to a failure to separate telomeres of sister chromatids. Hence, ctf18▵ and mre11A470T act in different pathways on telomere substrates for multiple phenotypes. The mre11A470T cells also displayed a DNA damage response (DDR) at 15°C but not at 30°C while ctf18▵ mutants conferred a constitutive DDR activity. Both the 15°C DDR pattern and growth rate were reversible at 30°C and displayed telomerase activity in vivo. We hypothesize that Ctf18 confers protection against stalling and/or breaks at the replication fork in cells that either lack, or are compromised for, telomerase activity. This Ctf18-based function is likely to contribute another level to telomere size homeostasis.

Review: The lord of the rings: Structure and mechanism of the sliding clamp loader

“…The Ctf18‐RFC complex is a seven subunit assembly, with the Ctf18 protein replacing RFC1 at the A position and the Dcc1 and Ctf8 proteins acting as auxiliary factors . Loss of Ctf18‐RFC disrupts sister chromatid cohesion, the S‐phase checkpoint response and telomere stability . Both Ctf18‐RFC and PCNA bind to the enzyme Eco1, which acetylates the Smc3 subunit of cohesin rings as a key step in establishment of sister chromatid cohesion .…”

Genetic instability in budding and fission yeast—sources and mechanisms

“… 2011 ) to maintain the telomeres (Gao et al. 2014 ) and is also involved in preserving proper genome copy number in the cell during division (Alabrudzinska, Skoneczny and Skoneczna 2011 ).…”

The many types of heterogeneity in replicative senescence

“…Because replication through the repetitive G‐rich telomeric sequences is difficult (Ivessa, Zhou, Schulz, Monson, & Zakian, ; Makovets, Herskowitz, & Blackburn, ; Miller, Rog, & Cooper, ; Sfeir et al, ), occasional replication errors at telomeres would fit the apparent stochastic nature of nonterminal arrests. This is supported by data that implicate multiple DNA and RNA processing enzymes and replication checkpoints in the onset of senescence (Azam et al, ; Balk et al, ; Fallet et al, ; Gao, Moss, Parke, Tatum, & Lustig, ; Grandin & Charbonneau, ; Lafuente‐Barquero et al, ), as discussed in Teixeira ().…”