Specific DNA Replication Mutations Affect Telomere Length in Saccharomyces cerevisiae

Abstract

To investigate the relationship between the DNA replication apparatus and the control of telomere length, we examined the effects of several DNA replication mutations on telomere length in Saccharomyces cerevisiae. We report that a mutation in the structural gene for the large subunit of DNA replication factor C (cdc44/rfc1) causes striking increases in telomere length. A similar effect is seen with mutations in only one other DNA replication gene: the structural gene for DNA polymerase alpha (cdc17/pol1) (M.J. Carson and L. Hartwell, Cell 42:249-257, 1985). For both genes, the telomere elongation phenotype is allele specific and appears to correlate with the penetrance of the mutations. Furthermore, fluorescence-activated cell sorter analysis reveals that those alleles that cause elongation also exhibit a slowing of DNA replication. To determine whether elongation is mediated by telomerase or by slippage of the DNA polymerase, we created cdc17-1 mutants carrying deletions of the gene encoding the RNA component of telomerase (TLC1). cdc17-1 strains that would normally undergo telomere elongation failed to do so in the absence of telomerase activity. This result implies that telomere elongation in cdc17-1 mutants is mediated by the action of telomerase. Since DNA replication involves transfer of the nascent strand from polymerase alpha to replication factor C (T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1950-1960, 1991; T. Tsurimoto and B. Stillman, J. Biol. Chem. 266:1961-1968, 1991; S. Waga and B. Stillman, Nature [London] 369:207-212, 1994), one possibility is that this step affects the regulation of telomere length.

CTC1‐STN1 coordinates G‐ and C‐strand synthesis to regulate telomere length

“…Introduction of dominant negative CTC1 A227V; V259M mutant into the CTC1 L1142H mutant background resulted in further telomere elongation, suggesting that CTC1 (and the CST complex) cooperates with DNA Pol‐α to negatively regulate telomerase. These findings are reminiscent of observations revealing that disrupting the interactions between CDC13 and DNA Pol‐α in yeast and CTC1 and DNA Pol‐α in mouse cells both result in telomere elongation (Adams & Holm, 1996; Chen et al., 2013; Grossi, Puglisi, Dmitriev, Lopes & Shore, 2004; Qi & Zakian, 2000). Our data also highlight the significant differences in telomere length maintenance mechanisms between normal and cancer cells.…”

Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C

“…Consistent with these data, we did not observe dramatic cell cycle delays in the asf1⌬ single-mutant cells, although the peaks were mildly broadened because of cell size heterogeneity. In contrast, rfc1-1 cells accumulated in the G2/M phase of the cell cycle, as previously reported (Adams and Holm 1996). The poorly growing asf1⌬ rfc1-1 mutant cells displayed a much more dramatic delay than the single-mutant cells, and the vast majority of cells existed in a single broad peak with approximately 2N DNA content.…”

“…rfc1-1 cells have elongated telomeres (Adams and Holm 1996), suggesting poor coordination of telomeric C-rich strand synthesis by the lagging strand machinery and G-rich strand synthesis by telomerase in these cells (Diede and Gottschling 1999). To rule out the possibility that hyperelongated telomeres were the cause of the Sphase delay in asf1⌬ rfc1-1 cells, telomere lengths were assayed.…”

Pol12, the B subunit of DNA polymerase α, functions in both telomere capping and length regulation

“…The original POL1 mutant described by Carson and Hartwell (1985) is a hypomorphic ts-lethal allele, presumably affecting the catalytic activity of the enzyme, that displays continual telomere elongation when grown at permissive or semipermissive temperatures, as well as elevated, RAD52-dependent recombination near telomeres. A more detailed examination of the pol1-1 telomere phenotype (Adams and Holm 1996;Adams Martin et al 2000) has revealed an S-phase-specific increase in singlestranded telomeric TG-repeat DNA, as well as a severe loss of telomeric silencing. Both of these phenotypes precede the telomere length increase seen after temperature up-shift, and are largely independent of telomerase action, suggesting that they are due to a primary defect in telomeric lagging-strand synthesis.…”

“…Point mutations in either protein that affect this interaction lead to a slight increase in average telomere length, but cause no other obvious phenotypes. However, mutations that affect the catalytic function of Pol1 and are lethal at elevated temperatures cause considerable and progressive telomere elongation, large increases in TG-specific single-stranded DNA at telomeres, and a loss of telomeric gene silencing (Carson and Hartwell 1985;Adams and Holm 1996;Adams Martin et al 2000). These and other data have led to the idea that Pol1, or the complete DNA polymerase ␣-primase complex, plays an important role in the down-regulation of telomerase action at telomeres, perhaps in a concerted regulatory step that ensures the replication of the CA-rich strand at chromosome ends.…”