COMMENTARY Genomic instability and repair mediated by common repeated sequences

Inbal Gazy and Martin Kupiec1 ITSs Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv A (TGTGTGGG)n 69978, Israel UR A3 TRP1 B UR A3 If you happen to find a suspicious cell, say, in event or a mutation occurs, the cells can be- TRP1 − your soup, you may recognize to what species come Ura and 5-FOA resistant. The rate of Stalled replication forks − DSB it belongs simply by looking at its chro- 5-FOA–resistant cells is in the order of 10 8 mosome configuration (karyotype) under the per division in a strain with a small intron at Cell death Regional changes in gene expression microscope. This approach works because URA3 Eror-free repair the gene. When 8 or 15 copies of the Point GCRs Tract mutations most cells of most organisms have a stable yeast telomeric sequences (TGTGTGGG) alterations genome, and gross chromosomal rearrange- were inserted within the intron, this rate in- C Possible GCRs A3 ments(GCRs)suchastranslocations,inver- creased 20- and 125-fold, respectively. Inter- Class 1) sions, and deletions of big chunks of estingly, in 75% and 38% of these, UR TRP1 chromosomes are relatively rare. This is in – U respectively, the 5-FOA resistant phenotype Class 2) stark contrast to cancer cells, which exhibit was due to point mutations affecting the cod- abundant GCRs (1). When the borders of ing capacity of the URA3 gene and not by Class 3) UR such rearrangements are mapped, they often gross amplifications/rearrangements. Thus, belong to repetitive sequences, which are the presence of telomeric repeats increases Class 4) scattered across the genome of all organisms. the rate of mutations in adjacent regions. UR Tandem repetitive sequences are also present The rest of the 5-FOA–resistant colonies car- at the ends of the eukaryotic chromosomes, ried complex chromosomal rearrangements Fig. 1. Genomic consequences of the presence of ITSs in forming the telomeres, which help replicate the genome. (A) Schematic description of the system used. (see below). Strikingly, small changes (addi- Either 8 or 15 copies of the telomere sequence TGTGTGGG the genome and protect it from degradation tions or deletions of a single repeat) within URA3 (2). Interestingly, telomeric repeats can also were inserted within an intron of the gene and placed the inserted telomeric sequences were very fre- on chromosome III. Triangles represent telomeric sequences; be found at internal positions along the chro- quent and could be detected even in the ab- circles represent centromeres; rhombs represent Ty elements mosomes in many organisms. These intersti- sence of selection: These events happened at (a yeast repeated sequence). (B) Schematic description of the tial telomeric sequences (ITSs) often colocalize −3 consequences of replication stalling at an ITS. (C)Fourtypes arateofabout10 per cell division. As ob- of gross chromosomal rearrangements described in the text. with chromosomal fragile sites and with end- served in other genomic regions that are hot- points of GCRs (3, 4). However, little is known In class 1, cells carry a terminal inversion; class 2 represents spots for genomic rearrangements, the high a repair event using URA3 and Ty sequences; and in classes 3 about the mechanisms responsible for genome rate of mutations and of small rearrangements and 4, a linear fragment coexists with a healed chromosome instability at interstitial telomeric sequences. suggest that double-strand breaks (DSBs) are that used internal Ty sequences (class 3) or Ty sequences in In PNAS, Aksenova et al. (5) present a study often created at the internal telomeric repeats; another chromosome (class 4) to heal. in which the power of yeast genetics is har- theirrepairisaccompaniedbyfrequentinser- nessed to address this particular question. Us- tions/deletions, mutations, and GCRs (Fig. i) Class 1 consisted of a large (80 kb long) in- ing a sophisticated genetic trap, the authors B version, in which the normal telomeric se- measure the rate of GCR formation and char- 1 ). This is in accordance with the fact that quences are now adjacent to half the URA3 acterize the molecular mechanisms leading to telomeric regions, being G-rich, are notori- ously hard to replicate, and tend to stall the gene, and the other half of the gene is now at them. The results show a surprisingly high C level of recombinational activity involving replication machinery, which can potentially thetelomere(Fig.1 ). Such a structure can be these repeated sequences. create DSBs (6). created by a DSB at the ITS, followed by re- section of the ends, and annealing/ligation in To investigate the frequency at which GCRs Analysis of GCRs Generated by the involving ITSs occur, Aksenova et al. use the inverse orientation. It is thus likely that in a clever yeast genetic trick (Fig. 1A): A yeast Presence of Internal Telomeric about half of the cases, when the annealing is strain carrying an intron-containing URA3 Sequences carried out in the normal orientation, such gene (involved in uracil biosynthesis) on chro- Almost half of the 5-FOA–resistant colonies events will stay undetected or only result in + mosome III is phenotypically Ura , because in the strain with 15 telomeric copies and gain/loss of a small number of telomeric re- + the intron is spliced efficiently. Ura cells can a quarter of those with 8 copies carried gross peats at the ITS. Interestingly, during the for- grow on plates lacking uracil, but cannot grow chromosomal rearrangements. These were mation of the rearranged structures, the on plates containing the toxic compound analyzed by a combination of pulse-field gel 5-fluoroorotic acid (5-FOA). Within this in- electrophoresis, PCR, comparative genome tron, they inserted an ITS-like sequence (com- hybridization (CGH), and sequencing and Author contributions: I.G. and M.K. wrote the paper. posed of telomere repeats), still enabling were found to fall into four distinct categories. The authors declare no conflict of interest. proper splicing and expression of the URA3 All of the events can be explained assuming See companion article on page 19866. fi gene. If the tandem repeats are ampli ed be- that a DSB took place during replication of the 1To whom correspondence should be addressed. E-mail: martin@ yond a certain size, or if, alternatively, a GCR internal telomeric sequences. post.tau.ac.il.

19664–19665 | PNAS | December 3, 2013 | vol. 110 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1320030110 Downloaded by guest on October 3, 2021 number of telomeric repeats increased from maintaining the stability of the genome. example, class 1 events, which can be ex- COMMENTARY 15 to 40–60, resulting in the silencing of Simple repeats, such as the ITSs (which may plained by a simple inversion, could be the adjacent TRP1 gene. These experiments themselves represent “scars” of previous mediated by nonhomologous end joining demonstrate how dynamic changes in chro- chromosomal rearrangements and repair (dependent on the ligase IV activity), by mosomal sequences may have profound con- events), present a challenge to the replication a recombinational mechanism requiring sequences for gene expression and thus for machinery and promote the breakage of strand invasion of duplex GT repeats by the phenotype and fitness of the cells. the telomeric ssDNA (which should re- ii)Class 2 was probably created by repair of Aksenova et al. show quire the activity of Rad51), or by a simple the two ends of the broken chromosome that repeated sequence annealing between resected ends [similar to by homologous recombination, using a sin- elements in the genome single strand annealing (SSA), and therefore gle template, the second copy of URA3 pres- Rad52 dependent but Rad51 independent]. ent at its normal location on chromosome V play important roles Other open questions to explore in the future (Fig. 1C). This ectopic recombination event, in both disrupting are whether telomere-affecting genes (12, 13) which creates a deletion in chromosome III, may play some role in preventing the type of was facilitated by the presence, within the and maintaining the rearrangements described. URA3 allele on chromosome V, of another stability of the genome. Conclusions type of repeated sequence, a Ty element. chromosomes (10). The cells use all of the The study of Aksenova et al. (5) provides These elements are retrotransposons that tricks available to try to repair the broken further evidence for the dynamic nature of change location by transposition at extremely chromosomes and to survive. Remarkably, genomes. The authors show that spontane- low levels (7) but are very actively involved in repetitive sequences also play a central ous lesions probably occur, at a single ITS, at − genomic rearrangements caused by homolo- role in the attempts to patch the broken a frequency above 10 3 per generation, and gous recombination (8, 9). With ∼35 copies chromosomes. this is a clear underestimate, as only events of Ty elements per haploid genome, and sev- As ITSs promote genomic instability, they thatintroduceavisiblechangecanbede- eral hundred copies of the 340-bp-long ter- must be under a strong evolutionary pressure tected in the current setting. If one multiplies minal repeats (LTRs), they represent a to change, so as not to create “trouble.” One this effect by the number of potential re- substantial fraction of the genome. way in which cells deal with this problem is to peated sequences with effects on the replica- iii) In Class 3, a linear, acentric fragment of change their chromatin configuration (4). tion efficiency, the total potential for genomic 80 kb is detected, and the centromere- Telomere position effect or variegation is instability is astounding, and it is remarkable containing fragment was healed by a common to all eukaryotic organisms, and how efficient the DNA damage response and break-induced replication (BIR) event that ITSs are also found in a heterochromatic repair systems of the WT cells are in pre- fi duplicated the intact distal chromosomal con guration (11). In this sense, the ITS an- venting genomic rearrangements. It is also end (Fig. 1C). Again, chromosome healing alyzed in the present paper might be partic- quite remarkable that repetitive genomic se- ularly active, as, being within an actively was accomplished by taking advantage of quences, which are the source of this in- expressed gene, it is probably present in Ty sequences with the right orientation in stability, are also part of its solution. a euchromatic context. It will be interesting the genome. to compare its behavior to that of a similar ACKNOWLEDGMENTS. Research in the M.K. labora- iv ) Class 4 is similar to class 3, with the differ- natural heterochromatic sequence. The ge- tory is supported by grants from the Israel Science ence that the Ty element used to heal the Foundation, the Israeli Ministry of Science and Technol- netic control of ITS-promoted rearrange- ogy, the Israel Cancer Fund, and the Israel Cancer broken centromere-containing chromosome ments is also extremely interesting: For Research Foundation. III was located on another chromosome, and thus a nonreciprocaltranslocationwas created (Fig. 1C). 1 Abeysinghe SS, Chuzhanova N, Cooper DN (2006) Gross 8 Mieczkowski PA, Lemoine FJ, Petes TD (2006) Recombination deletions and translocations in human genetic disease. Genome between retrotransposons as a source of chromosome The 80-kb linear acentric plasmid carries Dyn 1:17–34. rearrangements in the yeast Saccharomyces cerevisiae. DNA Repair 2 Blackburn EH (2010) Telomeres and telomerase: The means to (Amst) 5(9-10):1010–1020. telomeric sequences at its ends (one natural the end (Nobel lecture). Angew Chem Int Ed Engl 49(41): 9 Kupiec M, Petes TD (1988) Allelic and ectopic recombination and one extended from the ITS in the URA3 7405–7421. between Ty elements in yeast. Genetics 119(3):549–559. intron) and is surprisingly stable, suggesting 3 Lin KW, Yan J (2008) Endings in the middle: Current knowledge of 10 Rivero MT, Mosquera A, Goyanes V, Slijepcevic P, Fernández JL interstitial telomeric sequences. Mutat Res 658(1-2):95–110. (2004) Differences in repair profiles of interstitial telomeric sites that a partitioning mechanism able to segregate 4 Ruiz-Herrera A, Nergadze SG, Santagostino M, Giulotto E (2008) between normal and DNA double-strand break repair deficient it correctly between mother and daughter cells Telomeric repeats far from the ends: Mechanisms of origin and role in Chinese hamster cells. Exp Cell Res 295(1):161–172. may exist. evolution. Cytogenet Genome Res 122(3-4):219–228. 11 Ottaviani A, Gilson E, Magdinier F (2008) Telomeric position 5 Aksenova AY, et al. (2013) Genome rearrangements caused by effect: From the yeast paradigm to human pathologies? Biochimie Repeated Sequences Break, Repeated interstitial telomeric sequences in yeast. Proc Natl Acad Sci USA 90(1):93–107. 110:19866–19871. 12 Askree SH, et al. (2004) A genome-wide screen for Sequences Repair 6 Anand RP, et al. (2012) Overcoming natural replication barriers: Saccharomyces cerevisiae deletion mutants that affect telomere The results of Aksenova et al. (5) show that Differential helicase requirements. Nucleic Acids Res 40(3): length. Proc Natl Acad Sci USA 101(23):8658–8663. 1091–1105. 13 Ungar L, et al. (2009) A genome-wide screen for essential yeast repeated sequence elements in the genome 7 Boeke JD, Garfinkel DJ, Styles CA, Fink GR (1985) Ty elements genes that affect telomere length maintenance. Nucleic Acids Res play important roles in both disrupting and transpose through an RNA intermediate. Cell 40(3):491–500. 37(12):3840–3849.

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