And C Strand Length During New Telomere Synthesis

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications -- Chemistry Department Published Research - Department of Chemistry November 1997 Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis Xinqing Fan University of Nebraska - Lincoln Carolyn Mary Price University of Nebraska - Lincoln Follow this and additional works at: https://digitalcommons.unl.edu/chemfacpub Part of the Chemistry Commons Fan, Xinqing and Price, Carolyn Mary, "Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis" (1997). Faculty Publications -- Chemistry Department. 14. https://digitalcommons.unl.edu/chemfacpub/14 This Article is brought to you for free and open access by the Published Research - Department of Chemistry at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications -- Chemistry Department by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Molecular Biology of the Cell Vol. 8, 2145–2155, November 1997 Coordinate Regulation of G- and C Strand Length during New Telomere Synthesis Xinqing Fan and Carolyn Mary Price* Departments of Chemistry and Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 Submitted June 23, 1997; Accepted August 25, 1997 Monitoring Editor: Joseph Gall 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 pola and pold, our results suggest that this coordinate length regulation is mediated by DNA polymerase. INTRODUCTION effect is observed in yeast where mutations in telomerase components or telomere proteins lead to telo- In most organisms telomere length is determined by a mere shortening and senescence (Singer and balance between activities that elongate and shorten Gottschling, 1994; Nugent et al., 1996; Virta-Pearlman the telomeric DNA (Greider, 1996). Telomere shorten- et al., 1996). ing is caused by incomplete replication of the 59 end of Genetic experiments in yeast have shown that telo- the chromosome or by nuclease action, whereas elon- mere length regulation is a complex process that in- gation is caused either by telomerase, the specialized volves many different molecules. For example, telo- terminal transferase that extends the G-rich strand of mere length is affected by mutations not only in the telomere, or by recombination. The balance be- telomerase, but also in telomere-binding proteins, the tween telomere lengthening and shortening is ex- DNA replication machinery, helicases, and check tremely important for long-term cell viability as con- point-related proteins (Carson and Hartwell, 1985; tinuous telomere lengthening or shortening results in Schulz and Zakian, 1994; Singer and Gottschling, 1994; increased cell death (Lundblad and Szostak, 1989; Morrow et al., 1995; Zakian, 1995; Adams and Holm, McEachern and Blackburn, 1995; Krauskopf and 1996; Nugent et al., 1996; Virta-Pearlman, 1996; Cooper Blackburn, 1996; Wright et al., 1996). For example, in et al., 1997). One general theme to emerge is that human somatic cells the absence of telomerase leads to telomere proteins such as Rap1, Rif1, Rif2, Cdc13, and progressive loss of telomeric DNA at each population Est1 set up a complex chromatin structure that regu- doubling until the cells eventually become senescent lates telomerase access to the DNA terminus (Zakian, and die (Greider, 1996; Wright et al., 1996). A similar 1995; Krauskopf and Blackburn, 1996; Cooper et al., 1997; Marcand et al., 1997). This in turn determines * Corresponding author: Department of Chemistry, University of whether or not telomerase can add additional telo- Nebraska, Lincoln, Nebraska 68588. meric DNA. However, it is still unclear what role(s) © 1997 by The American Society for Cell Biology 2145 X. Fan and C.M. Price the checkpoint proteins, DNA pola, and replication novo telomere synthesis because this species will mate factor C play in the length regulation process. synchronously, thus allowing one to obtain cultures in It has been difficult to address the role of DNA which all the cells are at a particular stage of macro- polymerase in telomere length regulation as muta- nuclear development. tions in replication proteins usually affect general Telomere length is very precisely regulated in ma- chromosomal replication in addition to any telomeric ture Euplotes macronuclei as the G-strands are exactly functions. Thus, although mutations in yeast pola and 42 nucleotides long while the C-strands are 28 nucle- replication factor C cause telomere lengthening (Car- otides long. As a result, each telomere consists of 28 son and Hartwell, 1985; Adams and Holm, 1996), it is base pairs of C4A4zG4T4 duplex DNA and a 14-nucle- unclear whether these proteins are directly involved otide G strand overhang (Klobutcher et al., 1981). In in telomere length regulation or whether the muta- contrast, the G-strands of newly synthesized telo- tions cause a secondary effect on telomere length as a meres are heterogeneous in length and range from result of a general replication defect. It is widely as- 33–106 nucleotides. The median length is 94–95 nucle- sumed that DNA pola/primase is an active partici- otides (Vermeesch and Price, 1994). Surprisingly, the pant in telomere maintenance as the normal DNA C-strands of newly synthesized telomeres are much replication machinery is thought to generate the telo- less heterogeneous than the G-strands as the majority meric C-strand once telomerase has extended the G- are exactly 84 nucleotides long. Thus, C strand length strand (Greider, 1996; Skopp et al., 1996; Reveal et al., is much more tightly regulated than G strand length. 1997). However, there is little experimental evidence This finding suggests that although synthesis of the to support this contention. G-strand (by telomerase) is the first step in new telo- The ciliate Euplotes crassus has proved particularly mere addition, a major regulatory step occurs during useful for studying telomere biochemistry because subsequent C strand synthesis. this organism has literally millions of telomeres that Our discovery, that C- rather than G strand length is are generated by a multistep process during the sexual tightly regulated during new telomere addition, led us stage of the life cycle (Prescott, 1994). Since no general to wonder whether G- and C strand synthesis might DNA replication occurs during the new telomere syn- be regulated coordinately rather than by independent thesis, Euplotes provides a unique opportunity to mechanisms. Since the telomeric C-strand is thought study the role of DNA polymerase in telomeric C to be synthesized by DNA pol a/primase, we decided strand synthesis without the complication of simulta- to test for coordinate G- and C strand regulation by neous DNA replication. We have made use of this determining what happens to G strand length when C aspect of Euplotes biology to examine the link between strand synthesis is inhibited using a DNA polymerase C strand synthesis and telomere length regulation. inhibitor. We show here that if the drug aphidicolin is Like other ciliates, Euplotes has two structurally and used to partially inhibit DNA polymerase, both G- and functionally distinct nuclei: the germline micronu- C strand length are altered. These results indicate that cleus and the transcriptionally active macronucleus. telomeric G- and C strand synthesis are indeed coor- The micronucleus contains 50–100 large chromosomes dinately regulated. Our results also suggest that DNA whereas the macronucleus contains millions of linear polymerase is involved in this coordinate length reg- gene-sized DNA molecules [average size 2 kilobases ulation. (kb)] that have telomeres on each end (Prescott, 1994). The macronucleus is formed from a copy of the mi- MATERIALS AND METHODS cronucleus as a result of a complex genomic reorgani- zation that takes place when Euplotes cells mate (see Growth of E. crassus and Isolation of Anlagen DNA Figure 1 and Jahn, 1991). During this ;100-h process Euplotes cells were grown and mated as previously described (Price the DNA in the developing macronucleus (or anlage) 1990; Price et al., 1994) except that the cultures were concentrated first replicates to form polytene chromosomes (Figure fivefold just before aphidicolin addition. The timing of telomere addition was determined by Southern hybridization and

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