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Telomere Shortening Is Proportional to the Size of the G-rich Telomeric 3′-Overhang

Telomere Shortening Is Proportional to the Size of the G-rich Telomeric 3′-Overhang THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 26, Issue of June 30, pp. 19719 –19722, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Telomere Shortening Is Proportional to the Size of the G-rich Telomeric 3*-Overhang* Received for publication, April 4, 2000 Published, JBC Papers in Press, April 27, 2000, DOI 10.1074/jbc.M002843200 Kenneth E. Huffman‡, Stephen D. Levene‡, Valerie M. Tesmer§, Jerry W. Shay§, and Woodring E. Wright§¶ From the ‡Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083 and the §Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039 Most normal diploid human cells do not express telom- concept that this fill-in activity is carried out by the conven- tional lagging strand synthetic machinery (13). The 12–14- erase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the nucleotide single-stranded G-rich 39-overhang in hypotrichous length of the single-stranded G-rich telomeric 3*-over- ciliate telomeres (14) and the identification of a primase activ- hang is proportional to the rate of shortening in four ity that can initiate DNA synthesis at the very 39-end of the human cell types that exhibit different rates of telomere G-rich strand (15, 16) have led to the concept that the overhang shortening in culture. These results provide direct evi- is produced following digestion of a terminally positioned RNA dence that the size of the G-rich overhang is not fixed primer. Telomeres of yeast mutants lacking telomerase shorten but subject to regulation. The potential ability to manip- by only 3–5 bp per division (17), showing that even in the ulate this rate has profound implications both for slow- absence of telomerase yeast end-processing activities are able ing the rate of replicative aging in normal cells and for to replicate all but a few nucleotides at the end of the telomere. accelerating the rate of telomere loss in cancer cells in In contrast, rates of telomere shortening in human cells lacking combination with anti-telomerase therapies. telomerase can vary from 30 to several hundred bp per division (2–5). The length of the single-stranded G-rich telomeric overhang Telomerase is not expressed in most normal tissues but is in some human cells has been shown to be 150 –200 nucleotides present in 85–90% of all human tumors (1), and there is con- (18 –20), suggesting that either nuclease processing of the C- siderable interest in the potential oncologic use of telomerase rich strand is extensive or that the final RNA primer for lag- inhibitors. One concern is that such inhibitors would not di- ging strand synthesis is not placed at the very end of the G-rich rectly kill tumor cells but only initiate telomere shortening, strand. However, these studies did not determine whether this and thus it might take many cell divisions before a therapeutic length varied in cells whose telomeres shortened at different effect occurred. Cultured human cells exhibit different rates of rates. Variable rates of telomere loss could be due to processing telomere shortening (2–5), implying that this rate is not fixed events that affected both C- and G-rich strands, and that did but might be subject to manipulation. Agents that accelerate not change the size of this overhang, to different rates of sus- the rate of shortening might greatly augment the efficacy of taining oxidative damage that caused the loss of large seg- anti-telomerase treatments. However, virtually nothing is ments of telomeric DNA (21, 22) without affecting the size of known about what controls the rate of telomere shortening in the overhang on the remaining telomeres, or to mechanisms normal telomerase-negative human cells. that regulate the size of the single-stranded overhang. To dis- Telomeres of eukaryotic cells contain G-rich single-stranded tinguish between these possibilities, we examined the length of 39-overhangs, which extend beyond the double-stranded region. the single-stranded telomeric 39-overhang in human fibroblast, While the exact structure of these overhangs varies between breast epithelial, and vascular endothelial cell strains. We species, the presence of overhangs is both conserved and be- show that the size of the overhang is directly proportional to lieved to be essential for the maintenance of chromosome end the rate of telomere shortening, varying from about 300 nt in structure and function. Studies in ciliates and yeast indicate cells that lose 100 bp per division to a telomeric 39-overhang of that end-processing activities include 59-nucleases that digest 150 nt in cells that lose 50 bp per division. The size of the the C-rich telomeric strand, telomerase that elongates the G- overhang is thus an important correlate of the rate of telomere rich strand, nucleases that trim the G-rich strand so that it shortening (e.g. cells with long overhangs lose more telomeric ends at a nucleotide other than the normal telomerase pause repeats with each cell division). Possible mechanisms regulat- site, and activities that fill-in the C-rich strand (6 –11). DNA ing overhang length are discussed. polymerases a and d and primase are all required for telomer- ase activity in Saccharomyces cerevisiae (12), supporting the EXPERIMENTAL PROCEDURES DNA from cultured cells was prepared using modifications that per- mit the rapid processing of large numbers of samples (23), digested with * This work was supported by National Institutes of Health Grants a mixture of six different restriction enzymes with 4-base recognition AGO1228 (to W. E. W.) and GM47898 and GM55871 (to S. D. L.) and by sites, and analyzed on agarose gels. The size of the telomere restriction an American Cancer Society postdoctoral fellowship (to V. M. T.). The fragments was determined from PhosphorImager scans (Molecular Dy- costs of publication of this article were defrayed in part by the payment namics), using weighted mean calculations that normalize the signal of page charges. This article must therefore be hereby marked “adver- intensity relative to the size of each digestion product (2). tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Telomeres were purified by annealing a biotinylated C-rich oligonu- To whom correspondence should be addressed: Dept. of Cell Biol- cleotide to the G-rich telomeric overhangs (19). The overhangs were ogy, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9039. Tel.: 214-648-2933; Fax: 214-648-8696; E-mail: [email protected]. The abbreviations used are: bp, base pair(s); nt, nucleotide(s). This paper is available on line at http://www.jbc.org 19719 This is an Open Access article under the CC BY license. 19720 Telomere Shortening Is Proportional to the Overhang FIG.1. Rate of telomere shortening for four normal human diploid cell strains. BJ foreskin fibroblasts (kindly provided by James Smith, Baylor Univer- sity Medical Center), IMR90 lung fibro- blasts (ATCC number 186), HME31 hu- man mammary epithelial cells (24), and human umbilical vein endothelial cells (kindly provided by Nancy Marks, Uni- versity of Texas Southwestern Medical Center) were cultured throughout their proliferative lifespan. DNA was collected at multiple population doubling levels and analyzed on agarose gels. In many cases, the same DNA sample was run on several different gels. Values are given 6 1 S.E. FIG.3. The rate of telomere shortening is directly propor- tional to the size of the 3*-overhang. The data from Figs. 1 and 2 are compared with determine whether or not there is a significant relation- ship between the rate of telomere shortening and the size of the telo- meric G-rich single-stranded 39-overhang. The fact that the S.E. of the slope is only 10% of the value indicates the presence of a highly signif- FIG.2. Size distribution of telomeric overhangs for four nor- icant relationship. mal human diploid cell strains. Telomeres purified from the four cell strains described in Fig. 1 were examined by electron microscopy to determine the size of the single-stranded G-rich 39-overhang. T4 gp32 length of the protein-coated region to a series of standards of filaments in typical images are indicated by arrows. The number of known sizes using electron microscopy (19). Fig. 2 shows the molecules analyzed, N, the weighted mean overhang length, L , and avg distribution of overhang lengths observed for these cells. Fig. 3 the S.E. are given for each sample. Values for BJ fibroblasts are taken demonstrates a very strong linear relationship between the from Ref. 19. The first set of 69 molecules described in Ref. 19 from PD rate of telomere shortening and the average size of the telo- 87 cells (approximately three to five doublings prior to senescence) was eliminated from the present analysis because of the possibility that the meric overhang, with a slope of 0.31 6 0.03. size of the overhang might be different in slowly dividing near-senes- cent cells. Only the second analysis of 109 molecules from PD 21 BJ DISCUSSION fibroblasts is presented here, to have data comparable with that from These observations show that cell strains in which telomeres the other cell types. shorten twice as fast (e.g. umbilical vein endothelial cells lose 101 6 8 bp per division, whereas BJ fibroblasts lose 49 6 5bp coated with T4 gp32 single-stranded binding protein, and the length of per division: Fig. 1) have overhangs that are twice as long (e.g. the overhang measured as described previously (19). The measured umbilical vein endothelial cells have 322 6 14-nt overhangs lengths were converted to nt by reference to the measured length of whereas BJ fibroblasts have 156 6 7-nt overhangs: Fig. 2). This decorated plasmid DNAs containing single-stranded gaps or overhangs suggests that the size of the overhang may be important in of known lengths. determining the rate of shortening. These results imply that RESULTS alternate hypotheses for telomere shortening that would not Normal diploid human cells were cultured, and throughout affect the length of the overhang, such as oxidative damage to their proliferative lifespan multiple DNA samples were ana- telomeric DNA producing single and double-strand breaks (21), lyzed. Fig. 1 shows the progressive decrease in telomere restric- are unlikely to contribute significantly to the rate of telomere tion fragment length that occurred as a function of the number shortening, at least in human cells cultured under normal of population doublings. The rate of telomere shortening varied conditions. from 49 6 5to101 6 8 bp per division. Telomeres were then The demonstration that the size of the overhang is directly purified from these four cell types early in their lifespan, and proportional to the rate of shortening and that the size of the the G-rich, single-stranded overhangs were coated with T4 overhang is not a fixed value but subject to modification in gene-32 single-stranded binding protein (gp32). The length of different cells greatly increases the need to understand the the overhang can be determined by comparing the measured detailed mechanisms regulating this process. An explanation of Telomere Shortening Is Proportional to the Overhang 19721 these mechanisms will require knowledge of the size of the overhang generated by the end-replication problem on the lag- ging strand, nuclease processing events that might occur on G- and C-rich strands in both daughter duplexes, and DNA repair or other pathways that might fill in gaps left by processing events. None of these factors has yet been characterized for vertebrate telomeres. Fig. 4 presents one model to facilitate the interpretation of our results. We favor the interpretation that nuclease processing of the telomere produced by leading strand synthesis results in a relatively small G-rich 39-overhang, the inability of lagging strand synthesis to position the final RNA primer near the 39 terminus results in a much larger 39-over- hang, and it is the size of this larger overhang that is the primary determinant of the rate of telomere shortening. An understanding of the underlying mechanism could lead to methods that increase the rate of shortening as an adjunct to anti-telomerase therapies for the treatment of cancer. In addi- tion, methods that slow the rate of shortening could be used to increase the replicative lifespan of cells both in vitro and in vivo. Acknowledgments—We thank M. Liao and B. Frank for excellent technical assistance. APPENDIX The following is an explanation of why the measured over- hang is likely to be a result of the failure to detect overhangs FIG.4. Model for telomere shortening. The quantitative relation- that are less than 50 nt in size. ship between the size of the overhang and rates of shortening are shown Let X and Y be the lengths of lagging- and leading-strand for one possible model of the factors that might produce single-stranded overhangs, respectively (Fig. 4). The rate of shortening, r,is overhangs. The model is shown for telomerase negative normal human given as as follows (Equation 1). cells, so the addition of telomeric sequences by telomerase is not con- sidered. X represents the final size of the overhang on the daughter X 1 Y molecule produced by lagging strand synthesis. As drawn, it represents r 5 (Eq. 1) the distance between the last RNA priming event of lagging-strand 4 synthesis and the end of the chromosome, but it could also be produced by nuclease digestion without altering the calculations. Y represents We find from the linear relationship between the observed the size of the overhang produced by nuclease processing (6 –10) of the rate of shortening and the measured overhang length, L app parental C-rich strand that templated leading strand synthesis. We that r ' L /3 (Fig. 3). app have not included possible contributions of nuclease digestion of the If the measured overhang length is correct, then the average G-rich 39-overhang in this model. If G-strand-specific nuclease activity measured overhang length will be weighted by the proportion were a prominent factor, it would result in the failure of the rate of shortening to be proportional to the size of the overhang. The formula of lagging- and leading-strand overhangs recovered. Let the for the rate of shortening from the above model is 0.25(X 1 Y). The ratio of lagging- to leading-strand overhangs be denoted by a. average overhang length is the average of the overhang in both daugh- is then given as follows (Equation 2). app ter strands, which is (X 1 Y) 4 2. Since 0.25(X 1 Y) 5 0.5[(X 1 Y) 4 2], the rate of shortening should be one-half the average overhang length. Y 1 aX The slope of the line in Fig. 3 demonstrates that the observed rate of L 5 (Eq. 2) app 1 1 a shortening is one-third of the size of the average measured overhang. Within the context of this model, this implies that the average meas- Combining Equations 1 and 2, we obtain the following ured overhang does not reflect the true average of all X and Y over- hangs. The average measured overhang length may differ from the true (Equation 3). average for the following reasons. 1) Telomeres were purified by an- nealing a biotinylated (CCCTAA) to restriction-digested double- X 1 Y Y 1 aX 4–6 5 (Eq. 3) stranded DNA and then retrieving telomeres in which the G-rich 39- 4 3~1 1 a! overhang had hybridized to the probe. Only 20 –50% of the telomeres are recovered using this procedure (19). Telomeres with very short Solving for a gives the following (Equation 4). overhangs, overhangs annealed within T-loop structures (25) and over- hangs inaccessible to hybridization due to non-canonical structures 3X 2 Y a 5 (Eq. 4) such as G-quartets might all contribute to the failure to retrieve all of X 2 3Y the telomeres. 2)Bromodeoxyuridine labeling experiments following a single round of replication indicate that the overhangs on the daughter Since a is a positive quantity, a value 3Y , X is consistent DNA molecules are not identical; telomeres with labeled C-rich strands with the observation that longer overhangs are recovered more are recovered two to three times more efficiently than telomeres with labeled G-rich strands (23). This suggests that the telomere with the efficiently than very short overhangs. newly synthesized C-rich strand generated by lagging-strand synthesis has a longer overhang than the telomere that is the product of leading strand synthesis (overhang X is thus drawn as being longer than overhang Y). Since our method for purifying telomeres enriches for the base molecules containing telomeric overhangs as small as 12 nt (19), we estimate the limits of detection of T4 gp32-decorated overhangs by products of lagging-strand synthesis (telomeres with X overhangs), our measured average overhang will not correspond to the true average of electron microscopy to be about 50 nt using our procedure. One possi- bility is that the slope of 0.31 is obtained because most of the X all X and Y overhangs. However, calculations attempting to explain the slope of 0.31 based on unequal recovery of X and Y overhangs yield overhangs are near or below the 50 nt limit of T4 gp 32 coated single- stranded regions that we are able to detect. Most of these ends may thus unreasonable values (10 –20-fold enrichment for the products of lagging strand synthesis rather than the 2–3-fold enrichment that we obtained be excluded from the calculation of the measured average overhang. This is roughly consistent with our ability to observe overhangs on only in previous experiments (23)) for the relative recovery of these ends (see “Appendix” for detailed calculations). 3) Although we can purify 5-kilo- about 70% of the purified telomeres (19). 19722 Telomere Shortening Is Proportional to the Overhang Harley, C. B. & Bacchetti, S. (1992) EMBO J. 11, 1921–1929 Larger values of Y require stronger biases for the recovery of 4. Shay, J. W., Wright, W. E., Brasiskyte, D. & Van der Haegen, B. A. (1993) lagging-strand over leading-strand overhangs than those we Oncogene 8, 1407–1413 observe. For example, taking Y 5 X/4 gives a value of 11 for a, 5. Vaziri, H., Schachter, F., Uchida, I., Wei, L., Zhu, X., Effros, R., Cohen, D. & Harley, C. B. (1993) Am. J. Hum. Genet. 52, 661– 667 which is significantly greater than the ratio of 2 to 3 that we 6. Wellinger, R. J., Wolf, A. J. & Zakian, V. A. (1993) Cell 72, 51– 60 have observed by using bromodeoxyuridine labeling (12). 7. Wellinger, R. J., Ethier, K., Labrecque, P. & Zakian, V. A. (1996) Cell 85, 423– 433 Relative sizes of X and Y that are roughly compatible with 8. Lingner, J., Cooper, J. P. & Cech, T. R. (1995) Science 269, 1533–1534 the 2–3-fold enrichment of lagging- over leading-strand over- 9. Garvik, B., Carson, M. & Hartwell, L. (1995) Mol. Cell. Biol. 15, 6128 – 6138 hangs are inconsistent with the observed distributions of over- 10. Nugent, C. I., Hughes, T. R., Lue, N. F. & Lundblad, V. (1996) Science 274, 249 –252 hang lengths. For example, Y 5 X/10 gives a 5 4.1, but creates 11. Greider, C. W. (1996) Annu. Rev. Biochem. 65, 337–365 other problems. From Fig. 4, r 5 0.25 (X 1 Y) and if Y 5 X/10, 12. Diede, S. J. & Gottschling, D. E. (1999) Cell 99, 723–733 then r 5 0.28X. In BJ cells, r is 49 bp per division, thus X 5 175 13. Price, C. M. (1997) Biochemistry (Mosc.) 62, 1216 –1223 14. Klobutcher, L. A., Swanton, M. T., Donini, P. & Prescott, D. M. (1981) Proc. nt and Y is 18 nt. However, no overhangs this short were Natl. Acad. Sci. U. S. A. 78, 3015–3019 detected (see Fig. 2), because we believe the limits of detection 15. Zahler, A. M. & Prescott, D. M. (1988) Nucleic Acids Res. 16, 6953– 6972 using T4 gp32 coating are about 50 nt. 16. Zahler, A. M. & Prescott, D. M. (1989) Nucleic Acids Res. 17, 6299 – 6317 17. Zakian, V. A. (1995) Science 270, 1601–1607 Using the value Y 5 X/4 gave an unreasonable value for a as 18. Makarov, V. L., Hirose, Y. & Langmore, J. P. (1997) Cell 88, 657– 666 described above. However, if this value is used to calculate X 19. Wright, W. E., Tesmer, V. M., Huffman, K. E., Levene, S. D. & Shay, J. W. (1997) Genes Dev. 11, 2801–2809 for BJ cells, we obtain r 5 1.25X, and hence X 5 39.2 nt. This 20. McElligott, R. & Wellinger, R. J. (1997) EMBO J. 16, 3705–3714 size is still below our estimated limit of detection. Thus, we 21. von Zglinicki, T., Saretzki, G., Docke, W. & Lotze, C. (1995) Exp. Cell Res. 220, conclude that the reason the apparent average overhang length 186 –193 22. Saretzki, G., Sitte, N., Merkel, U., Wurm, R. E. & von Zglinicki, T. (1999) is not equivalent to actual average is that Y is likely to be below Oncogene 18, 5148 –5158 the limits of detection. 23. Wright, W. E., Tesmer, V. M., Liao, M. L. & Shay, J. W. (1999) Exp. Cell Res. 251, 492– 499 REFERENCES 24. Shay, J. W., Van der Haegen, B. A., Ying, Y. & Wright, W. E. (1993) Exp. Cell 1. Shay, J. W. & Bacchetti, S. (1997) Eur. J. Cancer 5, 787–791 Res. 209, 45–52 25. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H. 2. Harley, C. B., Fletcher, A. B. & Greider, C. W. (1990) Nature 345, 458 – 460 3. Counter, C. M., Avilion, A. A., LeFeuvre, C. E., Stewart, N. G., Greider, C. W., & de Lange, T. (1999) Cell 97, 503–514 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Telomere Shortening Is Proportional to the Size of the G-rich Telomeric 3′-Overhang

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 26, Issue of June 30, pp. 19719 –19722, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Telomere Shortening Is Proportional to the Size of the G-rich Telomeric 3*-Overhang* Received for publication, April 4, 2000 Published, JBC Papers in Press, April 27, 2000, DOI 10.1074/jbc.M002843200 Kenneth E. Huffman‡, Stephen D. Levene‡, Valerie M. Tesmer§, Jerry W. Shay§, and Woodring E. Wright§¶ From the ‡Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083 and the §Department of Cell Biology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039 Most normal diploid human cells do not express telom- concept that this fill-in activity is carried out by the conven- tional lagging strand synthetic machinery (13). The 12–14- erase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the nucleotide single-stranded G-rich 39-overhang in hypotrichous length of the single-stranded G-rich telomeric 3*-over- ciliate telomeres (14) and the identification of a primase activ- hang is proportional to the rate of shortening in four ity that can initiate DNA synthesis at the very 39-end of the human cell types that exhibit different rates of telomere G-rich strand (15, 16) have led to the concept that the overhang shortening in culture. These results provide direct evi- is produced following digestion of a terminally positioned RNA dence that the size of the G-rich overhang is not fixed primer. Telomeres of yeast mutants lacking telomerase shorten but subject to regulation. The potential ability to manip- by only 3–5 bp per division (17), showing that even in the ulate this rate has profound implications both for slow- absence of telomerase yeast end-processing activities are able ing the rate of replicative aging in normal cells and for to replicate all but a few nucleotides at the end of the telomere. accelerating the rate of telomere loss in cancer cells in In contrast, rates of telomere shortening in human cells lacking combination with anti-telomerase therapies. telomerase can vary from 30 to several hundred bp per division (2–5). The length of the single-stranded G-rich telomeric overhang Telomerase is not expressed in most normal tissues but is in some human cells has been shown to be 150 –200 nucleotides present in 85–90% of all human tumors (1), and there is con- (18 –20), suggesting that either nuclease processing of the C- siderable interest in the potential oncologic use of telomerase rich strand is extensive or that the final RNA primer for lag- inhibitors. One concern is that such inhibitors would not di- ging strand synthesis is not placed at the very end of the G-rich rectly kill tumor cells but only initiate telomere shortening, strand. However, these studies did not determine whether this and thus it might take many cell divisions before a therapeutic length varied in cells whose telomeres shortened at different effect occurred. Cultured human cells exhibit different rates of rates. Variable rates of telomere loss could be due to processing telomere shortening (2–5), implying that this rate is not fixed events that affected both C- and G-rich strands, and that did but might be subject to manipulation. Agents that accelerate not change the size of this overhang, to different rates of sus- the rate of shortening might greatly augment the efficacy of taining oxidative damage that caused the loss of large seg- anti-telomerase treatments. However, virtually nothing is ments of telomeric DNA (21, 22) without affecting the size of known about what controls the rate of telomere shortening in the overhang on the remaining telomeres, or to mechanisms normal telomerase-negative human cells. that regulate the size of the single-stranded overhang. To dis- Telomeres of eukaryotic cells contain G-rich single-stranded tinguish between these possibilities, we examined the length of 39-overhangs, which extend beyond the double-stranded region. the single-stranded telomeric 39-overhang in human fibroblast, While the exact structure of these overhangs varies between breast epithelial, and vascular endothelial cell strains. We species, the presence of overhangs is both conserved and be- show that the size of the overhang is directly proportional to lieved to be essential for the maintenance of chromosome end the rate of telomere shortening, varying from about 300 nt in structure and function. Studies in ciliates and yeast indicate cells that lose 100 bp per division to a telomeric 39-overhang of that end-processing activities include 59-nucleases that digest 150 nt in cells that lose 50 bp per division. The size of the the C-rich telomeric strand, telomerase that elongates the G- overhang is thus an important correlate of the rate of telomere rich strand, nucleases that trim the G-rich strand so that it shortening (e.g. cells with long overhangs lose more telomeric ends at a nucleotide other than the normal telomerase pause repeats with each cell division). Possible mechanisms regulat- site, and activities that fill-in the C-rich strand (6 –11). DNA ing overhang length are discussed. polymerases a and d and primase are all required for telomer- ase activity in Saccharomyces cerevisiae (12), supporting the EXPERIMENTAL PROCEDURES DNA from cultured cells was prepared using modifications that per- mit the rapid processing of large numbers of samples (23), digested with * This work was supported by National Institutes of Health Grants a mixture of six different restriction enzymes with 4-base recognition AGO1228 (to W. E. W.) and GM47898 and GM55871 (to S. D. L.) and by sites, and analyzed on agarose gels. The size of the telomere restriction an American Cancer Society postdoctoral fellowship (to V. M. T.). The fragments was determined from PhosphorImager scans (Molecular Dy- costs of publication of this article were defrayed in part by the payment namics), using weighted mean calculations that normalize the signal of page charges. This article must therefore be hereby marked “adver- intensity relative to the size of each digestion product (2). tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Telomeres were purified by annealing a biotinylated C-rich oligonu- To whom correspondence should be addressed: Dept. of Cell Biol- cleotide to the G-rich telomeric overhangs (19). The overhangs were ogy, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9039. Tel.: 214-648-2933; Fax: 214-648-8696; E-mail: [email protected]. The abbreviations used are: bp, base pair(s); nt, nucleotide(s). This paper is available on line at http://www.jbc.org 19719 This is an Open Access article under the CC BY license. 19720 Telomere Shortening Is Proportional to the Overhang FIG.1. Rate of telomere shortening for four normal human diploid cell strains. BJ foreskin fibroblasts (kindly provided by James Smith, Baylor Univer- sity Medical Center), IMR90 lung fibro- blasts (ATCC number 186), HME31 hu- man mammary epithelial cells (24), and human umbilical vein endothelial cells (kindly provided by Nancy Marks, Uni- versity of Texas Southwestern Medical Center) were cultured throughout their proliferative lifespan. DNA was collected at multiple population doubling levels and analyzed on agarose gels. In many cases, the same DNA sample was run on several different gels. Values are given 6 1 S.E. FIG.3. The rate of telomere shortening is directly propor- tional to the size of the 3*-overhang. The data from Figs. 1 and 2 are compared with determine whether or not there is a significant relation- ship between the rate of telomere shortening and the size of the telo- meric G-rich single-stranded 39-overhang. The fact that the S.E. of the slope is only 10% of the value indicates the presence of a highly signif- FIG.2. Size distribution of telomeric overhangs for four nor- icant relationship. mal human diploid cell strains. Telomeres purified from the four cell strains described in Fig. 1 were examined by electron microscopy to determine the size of the single-stranded G-rich 39-overhang. T4 gp32 length of the protein-coated region to a series of standards of filaments in typical images are indicated by arrows. The number of known sizes using electron microscopy (19). Fig. 2 shows the molecules analyzed, N, the weighted mean overhang length, L , and avg distribution of overhang lengths observed for these cells. Fig. 3 the S.E. are given for each sample. Values for BJ fibroblasts are taken demonstrates a very strong linear relationship between the from Ref. 19. The first set of 69 molecules described in Ref. 19 from PD rate of telomere shortening and the average size of the telo- 87 cells (approximately three to five doublings prior to senescence) was eliminated from the present analysis because of the possibility that the meric overhang, with a slope of 0.31 6 0.03. size of the overhang might be different in slowly dividing near-senes- cent cells. Only the second analysis of 109 molecules from PD 21 BJ DISCUSSION fibroblasts is presented here, to have data comparable with that from These observations show that cell strains in which telomeres the other cell types. shorten twice as fast (e.g. umbilical vein endothelial cells lose 101 6 8 bp per division, whereas BJ fibroblasts lose 49 6 5bp coated with T4 gp32 single-stranded binding protein, and the length of per division: Fig. 1) have overhangs that are twice as long (e.g. the overhang measured as described previously (19). The measured umbilical vein endothelial cells have 322 6 14-nt overhangs lengths were converted to nt by reference to the measured length of whereas BJ fibroblasts have 156 6 7-nt overhangs: Fig. 2). This decorated plasmid DNAs containing single-stranded gaps or overhangs suggests that the size of the overhang may be important in of known lengths. determining the rate of shortening. These results imply that RESULTS alternate hypotheses for telomere shortening that would not Normal diploid human cells were cultured, and throughout affect the length of the overhang, such as oxidative damage to their proliferative lifespan multiple DNA samples were ana- telomeric DNA producing single and double-strand breaks (21), lyzed. Fig. 1 shows the progressive decrease in telomere restric- are unlikely to contribute significantly to the rate of telomere tion fragment length that occurred as a function of the number shortening, at least in human cells cultured under normal of population doublings. The rate of telomere shortening varied conditions. from 49 6 5to101 6 8 bp per division. Telomeres were then The demonstration that the size of the overhang is directly purified from these four cell types early in their lifespan, and proportional to the rate of shortening and that the size of the the G-rich, single-stranded overhangs were coated with T4 overhang is not a fixed value but subject to modification in gene-32 single-stranded binding protein (gp32). The length of different cells greatly increases the need to understand the the overhang can be determined by comparing the measured detailed mechanisms regulating this process. An explanation of Telomere Shortening Is Proportional to the Overhang 19721 these mechanisms will require knowledge of the size of the overhang generated by the end-replication problem on the lag- ging strand, nuclease processing events that might occur on G- and C-rich strands in both daughter duplexes, and DNA repair or other pathways that might fill in gaps left by processing events. None of these factors has yet been characterized for vertebrate telomeres. Fig. 4 presents one model to facilitate the interpretation of our results. We favor the interpretation that nuclease processing of the telomere produced by leading strand synthesis results in a relatively small G-rich 39-overhang, the inability of lagging strand synthesis to position the final RNA primer near the 39 terminus results in a much larger 39-over- hang, and it is the size of this larger overhang that is the primary determinant of the rate of telomere shortening. An understanding of the underlying mechanism could lead to methods that increase the rate of shortening as an adjunct to anti-telomerase therapies for the treatment of cancer. In addi- tion, methods that slow the rate of shortening could be used to increase the replicative lifespan of cells both in vitro and in vivo. Acknowledgments—We thank M. Liao and B. Frank for excellent technical assistance. APPENDIX The following is an explanation of why the measured over- hang is likely to be a result of the failure to detect overhangs FIG.4. Model for telomere shortening. The quantitative relation- that are less than 50 nt in size. ship between the size of the overhang and rates of shortening are shown Let X and Y be the lengths of lagging- and leading-strand for one possible model of the factors that might produce single-stranded overhangs, respectively (Fig. 4). The rate of shortening, r,is overhangs. The model is shown for telomerase negative normal human given as as follows (Equation 1). cells, so the addition of telomeric sequences by telomerase is not con- sidered. X represents the final size of the overhang on the daughter X 1 Y molecule produced by lagging strand synthesis. As drawn, it represents r 5 (Eq. 1) the distance between the last RNA priming event of lagging-strand 4 synthesis and the end of the chromosome, but it could also be produced by nuclease digestion without altering the calculations. Y represents We find from the linear relationship between the observed the size of the overhang produced by nuclease processing (6 –10) of the rate of shortening and the measured overhang length, L app parental C-rich strand that templated leading strand synthesis. We that r ' L /3 (Fig. 3). app have not included possible contributions of nuclease digestion of the If the measured overhang length is correct, then the average G-rich 39-overhang in this model. If G-strand-specific nuclease activity measured overhang length will be weighted by the proportion were a prominent factor, it would result in the failure of the rate of shortening to be proportional to the size of the overhang. The formula of lagging- and leading-strand overhangs recovered. Let the for the rate of shortening from the above model is 0.25(X 1 Y). The ratio of lagging- to leading-strand overhangs be denoted by a. average overhang length is the average of the overhang in both daugh- is then given as follows (Equation 2). app ter strands, which is (X 1 Y) 4 2. Since 0.25(X 1 Y) 5 0.5[(X 1 Y) 4 2], the rate of shortening should be one-half the average overhang length. Y 1 aX The slope of the line in Fig. 3 demonstrates that the observed rate of L 5 (Eq. 2) app 1 1 a shortening is one-third of the size of the average measured overhang. Within the context of this model, this implies that the average meas- Combining Equations 1 and 2, we obtain the following ured overhang does not reflect the true average of all X and Y over- hangs. The average measured overhang length may differ from the true (Equation 3). average for the following reasons. 1) Telomeres were purified by an- nealing a biotinylated (CCCTAA) to restriction-digested double- X 1 Y Y 1 aX 4–6 5 (Eq. 3) stranded DNA and then retrieving telomeres in which the G-rich 39- 4 3~1 1 a! overhang had hybridized to the probe. Only 20 –50% of the telomeres are recovered using this procedure (19). Telomeres with very short Solving for a gives the following (Equation 4). overhangs, overhangs annealed within T-loop structures (25) and over- hangs inaccessible to hybridization due to non-canonical structures 3X 2 Y a 5 (Eq. 4) such as G-quartets might all contribute to the failure to retrieve all of X 2 3Y the telomeres. 2)Bromodeoxyuridine labeling experiments following a single round of replication indicate that the overhangs on the daughter Since a is a positive quantity, a value 3Y , X is consistent DNA molecules are not identical; telomeres with labeled C-rich strands with the observation that longer overhangs are recovered more are recovered two to three times more efficiently than telomeres with labeled G-rich strands (23). This suggests that the telomere with the efficiently than very short overhangs. newly synthesized C-rich strand generated by lagging-strand synthesis has a longer overhang than the telomere that is the product of leading strand synthesis (overhang X is thus drawn as being longer than overhang Y). Since our method for purifying telomeres enriches for the base molecules containing telomeric overhangs as small as 12 nt (19), we estimate the limits of detection of T4 gp32-decorated overhangs by products of lagging-strand synthesis (telomeres with X overhangs), our measured average overhang will not correspond to the true average of electron microscopy to be about 50 nt using our procedure. One possi- bility is that the slope of 0.31 is obtained because most of the X all X and Y overhangs. However, calculations attempting to explain the slope of 0.31 based on unequal recovery of X and Y overhangs yield overhangs are near or below the 50 nt limit of T4 gp 32 coated single- stranded regions that we are able to detect. Most of these ends may thus unreasonable values (10 –20-fold enrichment for the products of lagging strand synthesis rather than the 2–3-fold enrichment that we obtained be excluded from the calculation of the measured average overhang. This is roughly consistent with our ability to observe overhangs on only in previous experiments (23)) for the relative recovery of these ends (see “Appendix” for detailed calculations). 3) Although we can purify 5-kilo- about 70% of the purified telomeres (19). 19722 Telomere Shortening Is Proportional to the Overhang Harley, C. B. & Bacchetti, S. (1992) EMBO J. 11, 1921–1929 Larger values of Y require stronger biases for the recovery of 4. Shay, J. W., Wright, W. E., Brasiskyte, D. & Van der Haegen, B. A. (1993) lagging-strand over leading-strand overhangs than those we Oncogene 8, 1407–1413 observe. For example, taking Y 5 X/4 gives a value of 11 for a, 5. Vaziri, H., Schachter, F., Uchida, I., Wei, L., Zhu, X., Effros, R., Cohen, D. & Harley, C. B. (1993) Am. J. Hum. Genet. 52, 661– 667 which is significantly greater than the ratio of 2 to 3 that we 6. Wellinger, R. J., Wolf, A. J. & Zakian, V. A. (1993) Cell 72, 51– 60 have observed by using bromodeoxyuridine labeling (12). 7. Wellinger, R. J., Ethier, K., Labrecque, P. & Zakian, V. A. (1996) Cell 85, 423– 433 Relative sizes of X and Y that are roughly compatible with 8. Lingner, J., Cooper, J. P. & Cech, T. R. (1995) Science 269, 1533–1534 the 2–3-fold enrichment of lagging- over leading-strand over- 9. Garvik, B., Carson, M. & Hartwell, L. (1995) Mol. Cell. Biol. 15, 6128 – 6138 hangs are inconsistent with the observed distributions of over- 10. Nugent, C. I., Hughes, T. R., Lue, N. F. & Lundblad, V. (1996) Science 274, 249 –252 hang lengths. For example, Y 5 X/10 gives a 5 4.1, but creates 11. Greider, C. W. (1996) Annu. Rev. Biochem. 65, 337–365 other problems. From Fig. 4, r 5 0.25 (X 1 Y) and if Y 5 X/10, 12. Diede, S. J. & Gottschling, D. E. (1999) Cell 99, 723–733 then r 5 0.28X. In BJ cells, r is 49 bp per division, thus X 5 175 13. Price, C. M. (1997) Biochemistry (Mosc.) 62, 1216 –1223 14. Klobutcher, L. A., Swanton, M. T., Donini, P. & Prescott, D. M. (1981) Proc. nt and Y is 18 nt. However, no overhangs this short were Natl. Acad. Sci. U. S. A. 78, 3015–3019 detected (see Fig. 2), because we believe the limits of detection 15. Zahler, A. M. & Prescott, D. M. (1988) Nucleic Acids Res. 16, 6953– 6972 using T4 gp32 coating are about 50 nt. 16. Zahler, A. M. & Prescott, D. M. (1989) Nucleic Acids Res. 17, 6299 – 6317 17. Zakian, V. A. (1995) Science 270, 1601–1607 Using the value Y 5 X/4 gave an unreasonable value for a as 18. Makarov, V. L., Hirose, Y. & Langmore, J. P. (1997) Cell 88, 657– 666 described above. However, if this value is used to calculate X 19. Wright, W. E., Tesmer, V. M., Huffman, K. E., Levene, S. D. & Shay, J. W. (1997) Genes Dev. 11, 2801–2809 for BJ cells, we obtain r 5 1.25X, and hence X 5 39.2 nt. This 20. McElligott, R. & Wellinger, R. J. (1997) EMBO J. 16, 3705–3714 size is still below our estimated limit of detection. Thus, we 21. von Zglinicki, T., Saretzki, G., Docke, W. & Lotze, C. (1995) Exp. Cell Res. 220, conclude that the reason the apparent average overhang length 186 –193 22. Saretzki, G., Sitte, N., Merkel, U., Wurm, R. E. & von Zglinicki, T. (1999) is not equivalent to actual average is that Y is likely to be below Oncogene 18, 5148 –5158 the limits of detection. 23. Wright, W. E., Tesmer, V. M., Liao, M. L. & Shay, J. W. (1999) Exp. Cell Res. 251, 492– 499 REFERENCES 24. Shay, J. W., Van der Haegen, B. A., Ying, Y. & Wright, W. E. (1993) Exp. Cell 1. Shay, J. W. & Bacchetti, S. (1997) Eur. J. Cancer 5, 787–791 Res. 209, 45–52 25. Griffith, J. D., Comeau, L., Rosenfield, S., Stansel, R. M., Bianchi, A., Moss, H. 2. Harley, C. B., Fletcher, A. B. & Greider, C. W. (1990) Nature 345, 458 – 460 3. Counter, C. M., Avilion, A. A., LeFeuvre, C. E., Stewart, N. G., Greider, C. W., & de Lange, T. (1999) Cell 97, 503–514

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Published: Jun 1, 2000

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