Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha

Telomeres, the protein–DNA complexes at the termini of eukaryotic chromosomes, are vital for the preservation and complete replication of the genome (for reviews, see Blackburn 1991; Zakian 1995; Greider 1996). The past few years have seen explosive growth in our knowledge of telomere DNA-binding proteins and of telomerase, the enzyme that synthesizes telomeric DNA (for review, see Lingner and Cech 1998). Much less is known about the interaction between these two macromolecular complexes. The mechanisms of telomere–telomerase interaction provide the subject of the investigations presented here. Typically telomeric DNA consists of tandem repeats of a short sequence with the guanine-rich strand oriented 5′ → 3′ toward the chromosome terminus (e.g., Oxytricha telomeres consist of T4G4 repeats; Klobutcher et al. 1981). Although telomere length varies from species to species, the protrusion of the G-rich strand as a single-stranded overhang is a feature conserved among ciliated protozoa (for review, see Zakian 1989, 1995), yeast (Wellinger et al. 1993, 1996; Zakian 1996), and mammals (Makarov et al. 1997; McElligott and Wellinger 1997; Wright et al. 1997). Thus, despite differences in telomeric sequence and length between species, there may be similar functional mechanisms in telomere maintenance. In addition to the simple DNA repeats, a number of proteins play integral roles in telomere structure and function. Telomere proteins are of two types: those that bind the double-stranded portion of the telomere and those that bind the single-stranded telomeric overhang (for reviews, see Fang and Cech 1995a; Greider 1996; Brun et al. 1997). The best characterized single-stranded telomeric DNA-binding protein is that of the ciliated protozoan Oxytricha nova; the crystal structure of its complex with telomeric DNA has been solved recently (M. Horvath, V. Schweiker, J. Ruggles, J.M. Bevilacqua, and S.C. Schultz, unpubl.). The protein is a heterodimer consisting of a 56-kD α subunit and a 41-kD β subunit (Gottschling and Zakian 1986; Price and Cech 1987). Initial research revealed that the α subunit forms a specific complex with telomeric DNA, whereas the β subunit does not bind to DNA with sequence specificity by itself. The α and β subunits together bind tenaciously to telomeric DNA to form a stable ternary complex (Gray et al. 1991). The β subunit is capable of chaperoning the formation of G-quartet structures (Fang and Cech 1993a). G-quartets form monovalent cation-induced tetraplex DNA structures (Williamson 1994), which previously were shown to be poor substrates for telomerase (Zahler et al. 1991). The highly charged carboxyl domain of the β subunit mediates G-quartet structure formation in vitro (Fang and Cech, 1993a). The α · β heterodimer does not form on DNA folded into G-quartets, but rather requires an unfolded telomeric DNA substrate (Raghuraman and Cech 1990). One of the functions of the telomere is to act as a substrate for telomerase, the ribonucleoprotein that catalyzes the synthesis of telomeric DNA repeats (for reviews, see Blackburn 1992; Greider 1996; Lingner and Cech 1998). The regulation of telomere length and telomerase activity appear pivotal for cellular life span (Lundblad and Szostak 1989; Harley and Villeponteau 1995; Bodnar et al. 1998). Telomerase and single-stranded telomere DNA-binding proteins share substrate specificity (the telomeric DNA overhang). Both localize to the Oxytricha replication band during S-phase (Fang and Cech 1995b), and the telomere DNA-binding protein localizes behind telomerase in these analyses. Thus, it is thought that the telomere protein may bind to newly synthesized telomeres for their protection. A similar situation pertains in Euplotes crassus, with the interesting additional feature of a replication-specific version of the telomere protein (Skopp et al. 1996). The effect of the Oxytricha telomere DNA-binding proteins on telomerase activity was studied initially by Shippen et al. (1994). They reported that the ternary complex was a substrate for telomerase, although not as good a substrate as DNA free in solution. Because these experiments used native proteins that cannot be isolated in large quantities, the integrity of the reconstituted telomere protein–DNA complexes was difficult to evaluate. Moreover, the α · DNA complex, although initially characterized by nitrocellulose filter binding and dimethylsulfate (DMS) protection (Gray et al. 1991; Fang et al. 1993), had not been visualized as a discrete complex with the biological DNA substrate by gel electrophoresis. The present studies used recombinant Oxytricha telomere DNA-binding proteins in conjunction with native agarose gel electrophoresis (J.M. Bevilacqua and S.C.Schultz, unpubl.) to monitor telomere protein complex formation. The effect of telomere protein subunits on the synthesis of telomeric repeats by telomerase was then analyzed. Telomere DNA-binding protein subunits were found to inhibit telomerase by altering the telomeric DNA substrate accessibility and, therefore, may collaborate with telomerase to maintain telomere length. A model of telomere protein involvement in telomere synthesis and length regulation is presented.