Copyright 0 1992 by the Genetics Society of America The Structure and Evolutionof Subtelomeric Y‘ Repeats in Saccharomyces cerevisiae Edward J.Louis’ and James E. Haber Rosenstiel Center and Department of Biology, Brandeis University, Waltham, Massachusetts 02254-91 10 Manuscript received September 25, 199 1 Accepted for publication March 28, 1992 ABSTRACT The subtelomeric Y’ family of repeated DNA sequences in the yeast Saccharomyces cerevisiae is of unknown origin and function. Y’s vary in copy number and location among strains. Eight Y‘s, from two strains, were cloned and sequenced over the same 3.2-kb interval in order to assess the within- and between-strain variation as well as address their origin and function. One entireY’ sequence was reconstructed from two clones presented here and apreviously sequenced 833-bp region. It contains two large overlapping open reading frames (ORFs). The putative protein sequences have no strong homologies to any known proteins except for one region that has 27% identity with RNA helicases. RNA homologous to each ORF was detected. Comparison of the sequences revealed that the known long (Y’-L) and short (Y’-S) size classes, which coexist within cells, differ by several insertions and/or deletions within this region. The Y’-Ls from strain Y55 alsodiffer from those of strain YPl by several short deletions in the same region. Most of these deletions appear to have occurred between short (2-10 bp) direct repeats. The single base pair polymorphisms and the deletions are clustered in the first half of the interval compared. There is 0.30-1.13% divergence among Y’-Ls within a strain and 1.15-1.75% divergence between strains in the interval. This is similar to known unique sequence variation but contrasts with the 8-18% divergence among the adjacent subtelomeric repeats, X. Subsets of Y‘s exhibit concerted evolution; however, more than one variant appears to be maintained within strains. The observed sequence variation disrupts the first ORF in many Y’s while most of the second ORF including the putative helicase region is unaffected. The structure and distribution of the Y’ elements are consistent with having originated as a mobile element. However, they now appear to move via recombination. Recombination can account for the homogenization within subsets ofY’s but does not account for the maintenance of different variants. EPEATED sequences are found in all genomes. One class of repeated sequence families are those R They can be tandemlyarrayed and/ordispersed found adjacent to the telomeres of many eukaryotic and can exist in few to many thousands of copies [see chromosomes. These sequences are arranged in a ARNHEIM(1983) for review].Many repeated se- complex mosaic of different repeats. In general, no quences haveknown functions suchas rDNAs but function has been attributed to these sequences (ZAK- many others are of unknown function. There is a IAN 1989). No homologies are found among the general observation of concerted evolution within a known telomere associated sequences of different or- repeated sequence family. Within-population and - ganisms, in contrast to the functional and sequence speciesvariation among repeats is much less than homologies of the G-rich telomere repeats (ZAKIAN expected for independent evolution and is at variance with the observed divergence of sequences between 1989). Their origins are also unknown, though the populations and species (ARNHEIM1983; BALTIMORE line-like nature of telomere associated sequences in 1981 ; DOVER1982; LEIGHBROWN and ISH-HOROW- Drosophila is consistent with a mobile element origin ITZ 198 ;1 OHTA 1983; SELKERet al. 198 1; SLIGHTOM, (VALGEIRSD~TTIR,TRAVERSE and PARDUE 1990). BLECHLand SMITHIES1980). This concerted evolu- The chromosome end in Saccharomyces cerevisiae tion is usually attributed to recombination (including (see Figure 1) consistsof the functional telomere unequal sister chromatid exchange, reciprocal ex- which is comprised of (G,.sT), repeats. Adjacent to change and gene conversion)among members leading this sequence are up to 4 tandem copies of the Y’ to homogenization. Observed homogeneity could also element. This element is highly conserved (based on be due to the rapid turnover of sequences, via trans- restriction maps and hybridization) and is found at position, with loss of diverged copies via segregation some but not all chromosome ends (CHAN andTYE (SELKERet al. 198 1). 1983b; SZOSTAKand BLACKBURN1982; WALMSLEYet ’ Current address: Yeast Genetics, Institute of Molecular Medicine, John al. 1984). Internal to these is another less well defined Radcliffe Hospital, Oxford OX3 9DU, England. repeated element, X. X sequence is found in the sub- Genetics 1111: 559-574 uuly, 1992) 560 E. J. Louis and J. E. Haber pBR OPI SUP! I UUA3 2 kb c”---) X SA 8 R AX A I I 9YPI-LI FIGURE1 .-The chromosome end in yeast and pYPI-ARC I I XA SA 8 R P AXA the structureof the Y’ clones. The chromsome end inyeast consistsof telomere sequences (G1.>T), represented by 0, which are preceded by up to four tandem copies of Y’ (shaded). These are then preceded by other repeated elements including X X SA 8 R P AXA before sequences unique to that chromosome arm are reached. The telomere sequences are found 1 I pYPI-L3 between copies of Y’s and sometimes between Y’s and the adjacent X element. Characteristic restric- X S A B R P AXA tion sites include Xhol (X), Sal1 (S), Asp718 (A), BamHI (B), EcoRl (R) and Puul (P) which are shown for the nine clones. The two size classes of Y’s (Y‘- L and Y’S) differ in region between the Sal1 and XA 55 R P AXA EcoRI sites. Individual Y’s were marked with pBR322, SUP11 and URA3 using the sequence shown (striped). This Y’ sequence, used to mark I I pYP 1-5 1 the Y’s, from Puul to a Sac1 site originated from X SS R P AXA another strain (HOROWITZand HARER1984). Xhol I fragments of these marked Y’s were then cloned I directly into E. coli. The size and extent of the nine I I py55-s I clones are indicated and the regions sequenced and reported here are underlined. 8 S AS8 R P AXA a-/6kb/- I I pY55-LI X S SB R P AXA telomeric region of all chromosome ends. The GI.9T clones from a third strain (CHAN and TYE1983a). telomere sequences are sometimes found between X Additional types of Y’s not found in all strains include and Y’ and between tandem copies of Y’s (WALMSLEY a degenerate form (LOUISand HABER 1990b) found et al. 1984), however, not all X-Y’ junctions contain in strain Y55 that has the internal half but not the telomeresequence (LOUIS and HABER1991); E. J. telomere adjacent half of Y’ and an extra long form LOUISand J. E. Haber, in preparation). Internal to found in strain YPI (our unpublished observation) the X and Y’ elements are other repeated sequences that has anapparent insertion within 1 kb of the (E. J. LOUISand J. E. HABER,in preparation) which telomere. The Y’ elements vary in copy number and are finally preceded by sequencesunique to each location between strains and exhibit apparent con- particular chromosome arm. certed evolution based on restriction site and frag- To study the short andlong term effects of recom- ment length variation (LOUISand HABER 1990b). bination on a repeated sequence family, we have ex- Recombination involving individual Y’s was also amined the telomere associated repeated sequence, studied. Reciprocal exchanges between dispersed cop- Y’, in S. cerevisiae in detail. Copy number, location ies of y’s do not result in lethal rearrangements as and restriction site variation in two strains have been they simply exchange one telomere for another. Ec- characterized (LOUISand HABER1990b). The major- topic recombination (between copies at different ge- ity of Y’s fall into oneof two size classesfirst described nomic locations) was observed as well as the expansion by CHAN andTYE (1 983a). These will be designated of single copies into tandem arrays via unequal sister- here as long(Y’-L) and short(Y’-S) due to an apparent chromatid exchange (LOUISand HABER 1990a).Ac- insertion/deletion difference. These two size classes quisitions and losses of Y’s from individual chromo- coexist within strains with longs usually outnumbering some ends were also observed. These recombinational shorts. There are 19:6 and 7:3, longs:shorts, in the interactions were nonrandom in the sense thata two strains studied here and 7: I among independent marked element of one of the two known size classes, Yeast Subtelomeric Repeats 56 1 Y’-L and Y’-S, tended to recombine with other mem- a marked Y’ (LOUISand HABER1990a). pYPl-LP is the bers of its size class. Transposition could not account terminal Y’ of a tandem pair that resulted from a marked single Y’ from one end of chromosome XVI moving to the for the majority of events which were all consistent distal end of a resident single Y’ on another chromosome with beingrecombinational (LOUIS and HABER (ZX). The other two resulted from the recombinational 1990a). Transposition would result in a gain of a Y‘ transfer (either reciprocal exchange or gene conversion) of at the recipient location; however, in most cases there the marker sequences to unmarked Y’s at another chromo- was no change in Y’ copy number at therecipient end some. pYPl-L3 is the result of a recombination event be- tween a marked Y‘ (from chromosome V or VZZZ) and an (see DISCUSSION). The recombinationalinteractions unmarked single Y’ (at chromosome X). pYPl-L4 is the could account for the observed variation and distri- result of a recombination event between a marked Y’ (from bution of Y’s seen among different strains(LOUIS and chromosome V or VZZZ) and an unmarked single Y’ (at HABER1990a).