Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis

Abstract

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G-and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G-and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G-and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA pol␣ and pol␦, our results suggest that this coordinate length regulation is mediated by DNA polymerase. INTRODUCTIONIn most organisms telomere length is determined by a balance between activities that elongate and shorten the telomeric DNA (Greider, 1996). Telomere shortening is caused by incomplete replication of the 5Ј end of the chromosome or by nuclease action, whereas elongation is caused either by telomerase, the specialized terminal transferase that extends the G-rich strand of the telomere, or by recombination. The balance between telomere lengthening and shortening is extremely important for long-term cell viability as continuous telomere lengthening or shortening results in increased cell death (Lundblad and Szostak, 1989;McEachern and Blackburn, 1995;Krauskopf and Blackburn, 1996;Wright et al., 1996). For example, in human somatic cells the absence of telomerase leads to progressive loss of telomeric DNA at each population doubling until the cells eventually become senescent and die (Greider, 1996;Wright et al., 1996). A similar effect is observed in yeast where mutations in telomerase components or telomere proteins lead to telomere shortening and senescence (Singer and Gottschling, 1994;Nugent et al., 1996;Virta-Pearlman et al., 1996). Genetic experiments in yeast have shown that telomere length regulation is a complex process that involves many different molecules. For example, telomere length is affected by mutations not only in telomerase, but also in telomere-binding proteins, the DNA replication machinery, helicases, and check point-related proteins (Carson and Hartwell, 1985;Schulz and Zakian, 1994;Singer and Gottschling, 1994;Morrow et al., 1995;Zakian, 1995;Adams and Holm, 1996;Nugent et al., 1996;Virta-Pearlman, 1996;Cooper et al., 1997). One general theme to emerge is that telo...

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

“…The coordination of Pol␣-primase and telomerase function may be a highly conserved feature of telomere replication. For example, studies of the active telomereformation process in mating cells of the hypotrichous ciliate Euplotes crassus have demonstrated a tight linkage between Pol␣ (and/or ␦) action and telomerase addition, as well as a physical association between telomerase and primase (Fan and Price 1997;Ray et al 2002).…”

Dysfunction of Chromatin Assembly Factor 1 Induces Shortening of Telomeres and Loss of 45S rDNA inArabidopsis thaliana

“…In addition to the conventional DNA replication machinery, telomere length is maintained by telomerase (Greider and Blackburn, 1985) in a replication-dependent manner (Fan and Price, 1997;Diede and Gottschling, 1999;Marcand et al, 2000;Qi and Zakian, 2000;Ray et al, 2002). It is possible that under conditions of prolonged and/or incomplete replication and chromatin assembly at telomeres in fas mutants, the recruitment of telomerase may be impaired and/or the time slot for its action may be insufficient, resulting in telomere erosion despite the presence of standard telomerase activity in vitro ( Figure 6A).…”

Cdc13 both positively and negatively regulates telomere replication

“…Impairment of DNA polymerase ␣ results in elongated telomeres, as well as an increase in the extent of the terminal singlestrand extension of the G-rich strand (Carson and Hartwell 1985;Adams and Holm 1996;Fan and Price 1997;Adams Martin et al 2000). To examine whether the cdc13-5 mutant similarly affected the extent of single-stranded G-rich DNA, nondenaturing in-gel hybridization was used to examine chromosome termini.…”

“…Several recent studies indicate that disruption of C-strand DNA synthesis can impair telomere length control (for review, see Price 1997;Evans and Lundblad 2000). In the ciliate Euplotes, inhibition of DNA polymerases ␣ and ␦ during de novo telomere synthesis alters the length of the telomeric C strand and also causes an increase in the length and heterogeneity of the G strand (Fan and Price 1997). Similarly, propagation of S. cerevisiae strains defective for components of the lagging strand DNA replication machinery cause a telomerase-dependent increase in telomere length (Carson and Hartwell 1985;Adams and Holm 1996;Adams Martin et al 2000).…”