yces Tehwes Acquire Single-St- TG1-3 Tails Late in S Phase

Alexander J. Wolf,” and Virginia A. Zakian’t *Fred Hutchinson Cancer Research Center tand University of Washington Seattle, Washington 98104 Summary Saccharomyces teiomeres consist of - 300 bp of C,.& TGlms DNA. Nondenaturing Southern hybridization, ca- pable of detecting - 60 to - 300 bases of TGl.a DNA, revealed that yeast telomeres acquired and lost TGlo tails, the predicted intermediate in teiomere replica- tion, in a ceil cycle-dependent manner. TGer tails were also detected on the ends of a linear plasmid isolated from late S phase ceils. In addition, a nonlinear form of this pm Wang and Zakian, 1990a). The yeast telomere sequence is similar to that of most other eukaryotes in having clusters of G residues in the strand running 5’to 3’from the center toward the end of the DNA molecule (hereafter called the G strand). G-strand telomeric oligonucleotides, including TGls oligonucleotides, can form a variety of structures in vitro held together by non-Watson-Crick G-G base pairs (Henderson et al., 1987; Sundquist and Klug, 1989; Wil- liamson et al., 1989; Sen and Gilbert, 1990; Acevedo et al., 1991; Lustig, 1992). Telomeres are, thought to be essential for ensuring the complete replication of the chromosome. Conventional DNA polymerases require a primer, normally RNA, and can synthesize DNA only in the 5’ to 3’ direction. As a result, replication by a conventional DNA polymerase is expected to leave i gap of - 8-12 nt, the size of RNA primers in yeast (Newlon, 1988) and other eukaryotes, at the 5’ ends of newly replicated strands. The loss of sequence information resulting from removal of RNA prim- ers is predictecj to be slow but progressive. Presumably, the telomere provides a substrate for an alternative repli- cation mechanism that can compensate for this loss. The ribonucleoprotein telomerase, an activity that ex- tends G-strand telomeric oligonucleotides in the absence of a DNA template, has been identified in several ciliated protozoa and in HeLa cells (reviewed in Blackburn, 1992). Although telomerase has not been detected in S. cerevis- iae, the DNA sequence requirements for telomere forma- tion in yeast (Murray et al., 1988; Pluta and Zakian, 1989) are similar to the substrate requirements displayed by the Tetrahymena telomerase in vitro (Greider and Blackburn, 1985; Harrington and Greider, 1991). Telomere-telomere recombination occurs during the formation of new telo- meres in S. cerevisiae (Pluta and Zakian, 1989). Because this recombination proceeds by a gene conversion mecha- nism that results in a net increase in telomeric DNA (Wang and Zakian, 199Ob), it too could contribute to the process of telomere replication. In both the telomerase and gene conversion models of telomere replication, DNA termini are predicted to have transient extensions of the TGts strand (Zakian et al., 1990). Using nondenaturing Southern hybridization, we demonstrate that telomeres on yeast chromosomes and linear plasmids acquire TGls tails late in S phase. In addi- tion, using two-dimensional agarose gel electrophoresis, we demonstrate that the TG13 tails on linear plasmids iso- lated from late S phase cells can mediate stable telomere- telomere interactions. Results Nondenaturing Southern Hybridization Can Be Used to Detect Single-Stranded TGl.J DNA A Southern hybridization method in which DNA is never denatured was used to determine if yeast telomeres have TG1.3 tails. A 280 bp stretch of C1.&TG1.3 DNA cloned from an authentic yeast telomere was used to test the system. The tract of duplex C1.&TG1-3 DNA was positioned at the end of a restriction fragment in the same orientation that it would have if it were at the end of a chromosome (Figure 1 B). This DNA was treated for increasing amounts of time with T7 (gene 6) exonuclease, an exonuclease that re- moves bases from the 5’ end of a duplex DNA molecule (Kerr and Sadowski, 1972), to create molecules with TG1.3 tails (Figure 16). To measure the lengths of TGjJ tails, T7 exo- nuclease-digested DNA was treated with mung bean nuclease, a single strand-specific endonuclease, and the sizes of the treated fragments were determined by conven- tional Southern hybridization (Figure 1 D). The fragments with TG,.3 tails were also analyzed by nondenaturing Southern hybridization using a &A riboprobe (Figure 1 C). The 280 bp of duplex C1.&TG1.3 DNA did not hybrid- ize in the nondenaturing Southern hybridization, whereas TG,.3 tails of 65 to 280 bases were readily detected. This experiment also demonstrated that TG1.3 tails of -30

[1]  D. Lockshon,et al.  The arrest of replication forks in the rDNA of yeast occurs independently of transcription , 1992, Cell.

[2]  A. Lustig Hoogsteen G-G base pairing is dispensable for telomere healing in yeast. , 1992, Nucleic acids research.

[3]  C. Greider Telomerase is processive , 1991, Molecular and cellular biology.

[4]  T. Macke,et al.  The coherence of synthetic telomeres. , 1991, Nucleic acids research.

[5]  R. Wellinger,et al.  Effects of excess centromeres and excess telomeres on chromosome loss rates , 1991, Molecular and cellular biology.

[6]  W. L. Fangman,et al.  A yeast origin of replication is activated late in S phase , 1991, Cell.

[7]  A. Wolf,et al.  RAP1 protein interacts with yeast telomeres in vivo: Overproduction alters telomere structure and decreases chromosome stability , 1990, Cell.

[8]  V. Zakian,et al.  Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains , 1990, Molecular and cellular biology.

[9]  V. Zakian,et al.  Telomere telomere recombination provides an express pathway for telomere acquisition , 1990, Nature.

[10]  K. Runge,et al.  How does the end begin? Formation and maintenance of telomeres in ciliates and yeast. , 1990, Trends in genetics : TIG.

[11]  Aaron Klug,et al.  Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops , 1989, Nature.

[12]  T. Cech,et al.  Monovalent cation-induced structure of telomeric DNA: The G-quartet model , 1989, Cell.

[13]  C. Newlon,et al.  Time of replication of ARS elements along yeast chromosome III , 1989, Molecular and cellular biology.

[14]  K. Runge,et al.  Introduction of extra telomeric DNA sequences into Saccharomyces cerevisiae results in telomere elongation , 1989, Molecular and cellular biology.

[15]  V. Zakian,et al.  Recombination occurs during telomere formation in yeast , 1989, Nature.

[16]  R. Wellinger,et al.  Lack of positional requirements for autonomously replicating sequence elements on artificial yeast chromosomes. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[17]  V A Zakian,et al.  Structure and function of telomeres. , 1989, Annual review of genetics.

[18]  C. Newlon Yeast chromosome replication and segregation , 1988, Microbiological reviews.

[19]  A. Murray,et al.  Characterization of two telomeric DNA processing reactions in Saccharomyces cerevisiae , 1988, Molecular and Cellular Biology.

[20]  R. McCarroll,et al.  Time of replication of yeast centromeres and telomeres , 1988, Cell.

[21]  C. C. Hardin,et al.  Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine·guanine base pairs , 1987, Cell.

[22]  S. Henikoff,et al.  Conserved arrangement of nested genes at the Drosophila Gart locus. , 1987, Genetics.

[23]  J. Huberman,et al.  The in vivo replication origin of the yeast 2μm plasmid , 1987, Cell.

[24]  W. L. Fangman,et al.  The localization of replication origins on ARS plasmids in S. cerevisiae , 1987, Cell.

[25]  Drena D. Larson,et al.  Dynamics of telomere length variation in tetrahymena thermophila , 1987, Cell.

[26]  G. Wahl,et al.  Northern and Southern blots. , 1987, Methods in enzymology.

[27]  Carol W. Greider,et al.  Identification of a specific telomere terminal transferase activity in tetrahymena extracts , 1985, Cell.

[28]  J. Haber,et al.  Subtelomeric regions of yeast chromosomes contain a 36 base-pair tandemly repeated sequence. , 1984, Nucleic acids research.

[29]  T. Petes,et al.  Unusual DNA sequences associated with the ends of yeast chromosomes , 1984, Nature.

[30]  Jack W. Szostak,et al.  DNA sequences of telomeres maintained in yeast , 1984, Nature.

[31]  P. Schimmel,et al.  Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product. , 1984, The Journal of biological chemistry.

[32]  A. Feinberg,et al.  A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. , 1983, Analytical biochemistry.

[33]  Clarence S. M. Chan,et al.  Organization of DNA sequences and replication origins at yeast telomeres , 1983, Cell.

[34]  K. Murata,et al.  Transformation of intact yeast cells treated with alkali cations , 1983 .

[35]  R. W. Davis,et al.  Centromeric DNA from Saccharomyces cerevisiae. , 1982, Journal of molecular biology.

[36]  V. Zakian,et al.  Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA , 1982, Molecular and cellular biology.

[37]  J. Hartley,et al.  Nucleotide sequence of the yeast plasmid , 1980, Nature.

[38]  R. W. Davis,et al.  Eukaryotic DNA segments capable of autonomous replication in yeast. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[39]  D. Kowalski,et al.  Gene-sized pieces produced by digestion of linear duplex DNA with mung bean nuclease. , 1978, Biochemistry.

[40]  J. Sutcliffe,et al.  pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. , 1978, Nucleic acids research.

[41]  D. Kupfer,et al.  Control of Saccharomyces cerevisiae 2microN DNA replication by cell division cycle genes that control nuclear DNA replication. , 1977, Journal of molecular biology.

[42]  C. Newlon,et al.  Replication of Yeast Chromosomal DNA , 1974, Nature.

[43]  I. Lehman,et al.  THE DEOXYRIBONUCLEASES OF ESCHERICHIA COLI. V. ON THE SPECIFICITY OF EXONUCLEASE I (PHOSPHODIESTERASE). , 1964, The Journal of biological chemistry.