With a Highly Selected Subset of TGI 3 Repeats
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Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press New telomeres in yeast are initiated with a highly selected subset of TGI_ 3 repeats Kate M. Kramer and James E. Haber 1 Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02254-9110 USA The creation of new telomeres was studied by generating a site-specific double-strand break in diploid strains of Saccharomyces cerevisiae that are unable to carry out homologous recombination. New telomere formation occurred -1% of the time but only when (T2G4113 was present proximal to the break site. About half of the healing events occurred at a number of 1- to 9-bp G or G,T sequences located as far as 128 bp distal to the T2G4 repeats. Surprisingly, in 16 events at sites ending in GTGG, the first TGI_ 3 nucleotides added always included either an 11- or a 13-bp sequence {GTGTGGGTGTG or GTGTGTGGGTGTG), after which each new telomere diverged into a less ordered TGx_3 pattern. Moreover, at 75% of the remaining addition sites, these same 11- or 13-bp sequences were found overlapping the junction between the chromosomal primer and the newly added sequences. We propose that short G,T sequences near an organizing sequence such as (T2G4)13 can act as primers to pair with the template RNA of a telomerase and add new sequences that are complementary to that RNA. [Key Words: Broken chromosome repair; Saccharomyces cerevisiae; new telomere formation] Received August 18, 1993; revised version accepted September 27, 1993. The stability of linear chromosomes depends on the cally programmed phase of cellular development and dif- presence of telomeres at their ends. Telomeres function ferentiation. For example, the Tetrahymena telomerase both to ensure the complete replication of chromosome adds telomere repeats to the ends of fragmented chromo- ends and to protect them from degradation, recombina- somes during macronuclear development, at sites where tion, and fusion with other chromosomes (for review, see no telomeric repeats are present {Yu and Blackburn 1991). Blackbum 1992; Blackburn and Szostak 1984; Zakian Similar programmed telomere addition also occurs in 1989; Biessmann and Mason 1992). Most eukaryotic te- Ascaris {Mfiller et al. 1991). New telomeres can also be lomeres consist of G-rich simple repeat sequences at the formed to repair chromosomes broken by mechanical chromosome terminus, for example, T2G4, T4G 4 or rupture or other stress. McClintock {1941) first described T2AG 3. The conservation of telomere sequences among such "healing" of broken chromosomes, and similar evolutionarily diverse organisms suggests that they all events have been documented in yeast {McCusker and share a common structure and function. The mechanism Haber 1981; Haber and Thorburn 1984; Surosky et al. by which telomeric repeats are added onto a preexisting 1986; J/iger and Philippsen 1989; K~imper et al. 1989). array have been well documented in ciliates (Greider and Spontaneously truncated chromosomes have also been Blackburn 1985, 1987, 1989; Zahler and Prescott 1988; found in organisms as diverse as malaria (Pologe and Shippen-Lenz and Blackburn 1989} and in both mice and Ravetch 1988; Scherf and Mattei 1992) and humans humans (Morin 1991; Prowse et al. 1993). In these cases, {Wilkie et al. 1990). highly regular telomere repeats are added by an RNA- In S. cerevisiae new TGI_3 telomeric sequences are dependent DNA polymerase (telomerase) in which the 3' efficiently added onto transformed linear plasmids car- end of the preexisting telomere sequence acts as a primer tying heterologous telomere repeats from a variety of for the addition of new telomere repeats. It is not yet organisms (for review, see Zakian 1989; Biessmann and clear, however, whether the yeast Saccharomyces cere- Mason 1992). There is yet no direct evidence of telom- visiae telomere repeats, an irregular array of TGI_a se- erase activity in yeast; however, a strong candidate gene quences, are added by a telomerase mechanism. for the yeast telomerase has been described in the gene In some circumstances, telomeres are synthesized de EST1 (Lundblad and Szostak 1989). RAD52-independent novo. New telomeres can be created as part of a geneti- recombination has been proposed as an alternative mechanism for the addition and maintenance of yeast 1Corresponding author. TGI_a (Wang and Zakian 1990b; Biessmann and Mason GENES & DEVELOPMENT 7:2345-2356 © 1993 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/93 $5.00 2345 Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Kramer and Haber 1992). This suggestion is based on the observation of contains one of two specific 11- or 13-bp motifs. Finally, recombination between heterologous Tetrahymena the specific TGI__3 sequences that are added to a given T2G4 repeats and Oxytricha TaG 4 repeats on opposite site are strongly influenced by the DNA sequence at that ends of a newly introduced linear yeast artificial chro- site. mosome (Pluta and Zakian 1989; Wang and Zakian 1990a). On the basis of these results it has been argued that yeast chromosome ends may sometimes acquire te- Results lomeric TGI_3 de novo by a RAD52-independent recom- Generating terminally deleted chromosomes binational pathway. However, this model does not easily account for how new telomeres could be added on to The strategy that we have used is illustrated in Figure 1. nontelomeric sequences that frequently intervene be- A site-specific chromosome break is introduced at the tween heterologous telomere repeats on transformed lin- mating-type (MAT~ locus on chromosome III by galac- ear plasmids and the newly added TG~_3 (Murray et al. tose-regulated induction of the HO endonuclease gene 1988; Wang and Zakian 1990b). It has been argued that (Jensen and Herskowitz 1984; Connolly et al. 1988). In this may occur by a copy choice mechanism or slippage the absence of RAD52 function, this double-strand break during DNA synthesis (Wang and Zakian 1990b; Bless- (DSB) cannot be repaired by homologous recombination, mann and Mason 1992). either with the silent copy donor sequences HML or To learn more about the way that new telomere addi- HMR or with the MAT information on the opposite chro- tion occurs in yeast, we have developed a simple system mosome (Weiffenbach and Haber 1981). Diploids con- to generate specific chromosome breaks that can be re- taining terminally deficient, healed chromosomes can paired by de novo telomere formation. Our goal was to then be recovered by genetic screening. Ten kilobases examine the actual sequences added at new telomeres, to proximal to the break site, the broken chromosome car- establish whether these events were likely to have arisen ries the URA3 marker and 13 T2G4 repeats flanked on by telomerase activity or by recombination. Our se- the distal side by nontelomeric DNA. We reasoned that quence analysis of new telomere addition sites leads us if new telomere formation could not occur at any of the to propose that new TGI_ 3 sequences are added by the sequences in the 10-kb interval adjacent to the break action of a telomerase rather than by recombination. We site, the T2G 4 repeats would act as a possible "default" base our model on the following observations. First, new site. telomere sequences can be added in a RAD52 indepen- Diploids of strain PA104TxRS200L (Table 1) carrying dent manner to short stretches (1-9 bp) of G, T sequence terminally deficient chromosomes are expected to be located up to 100 bp distal to an essential (T2G4)13 orga- Ura3 + but Thr4-; therefore, strains with this phenotype nizing sequence. In some cases, the substrates for addi- were analyzed for their karyotype by contour-clamped tion are as short as a single G, GT, GG, or GTG. Second, homogeneous electric fields (CHEF) gel electrophoresis the junction at the chromosomal addition site frequently and Southern blot analysis (see Materials and methods). Figure 1. Generating a terminally deficient a chromosome III. Schematic diagrams of chromosome HI are shown. (A) The rad52 GAL::HO strain PA104T was marked by an insertion of URA3::(TTGGGGh3::pBR322, 10 kb HML LEU2 0 ~ MATer! ) THR4 ,,HMR induce DSB in proximal to the HO cut site. [In strains rad52 PAl 04T R XB MA T~x haploid YKK104BX1, YKK104BX2, and YKK104BX3 }~ lOKb the pBR322 sequences are replaced by BX1, BX2, and BX3 sequences (see Materials leu2 (RS200L) mate a-like LEU2 (YKK611) ~ MATe thr4 HMR and methods)]. A DSB was generated at HML r (Mater1- Mater2-) MATa on chromosome III in PA104T fol- cells to RS200L (RAD52) or lowing HO endonuclease expression from a YKK611 (rad52) GAL::HO plasmid. The DSB is degraded by MA Tc~ haploid extensive 5' ~ 3' exonuclease activity (White and Haber 1990) such that MATer B URA3 (T2G4)13 pBR322 H gene functions are disrupted. The degrada- HE............................. I GAL::HO tion of -400 bp of a sequences causes cells to become phenotypically Mata2- Matal- HML C~ MATct]" THR4 HMR induce DSB and exhibit an a-mating behavior (Weiffen- }-- lOKb I bach and Haber 1981). These a-mating cells in rad52/rad52 HML MATa*inc thr4 HMR MA ToEMA Ta- inc were then rescued by mating to either a A III diploid YKK910 RAD52 (RS200L) or a rad52 (YKK611) MATa haploid carrying the distal, recessive marker thr4 on chromosome Ill. (B) The DSB was induced in a MATa/MATa-inc diploid (YKK910) homozygous for rad52. In the YKKgl0 diploid, only the MATs locus can be cut by HO endonuclease (Weiffenbach et al. 1983). Silent mating-type cassettes HML and HMR are indicated. (B) BamHI; (S) SmaI; (H) HindlII; (X) XbaI; (R) EcoRI. 2346 GENES& DEVELOPMENT Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press New telomere formation in yeast Table 1.