Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms

Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms View ORCID Profile Fernando R. Rosas Bringas , Ziqing Yin , Yue Yao , Jonathan Boudeman , Sandra Ollivaud , View ORCID Profile Michael Chang doi: https://doi.org/10.1101/2024.04.11.589032 Fernando R. Rosas Bringas a European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fernando R. Rosas Bringas Ziqing Yin a European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yue Yao a European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jonathan Boudeman a European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sandra Ollivaud a European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael Chang a European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen , Groningen, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael Chang For correspondence: m.chang{at}umcg.nl Abstract Full Text Info/History Metrics Preview PDF Abstract Telomeric DNA sequences are difficult to replicate. Replication forks frequently pause or stall at telomeres, which can lead to telomere truncation and dysfunction. In addition to being at chromosome ends, telomere repeats are also present at internal locations within chromosomes, known as interstitial telomeric sequences (ITSs). These sequences are unstable and prone to triggering gross chromosomal rearrangements (GCRs). In this study, we quantitatively examined the effect of ITSs on GCR rate in Saccharomyces cerevisiae using a genetic assay. We find that GCR rate increases exponentially with ITS length. This increase can be attributed to the telomere repeat binding protein Rap1 impeding DNA replication and a bias of repairing DNA breaks at or distal to the ITS via de novo telomere addition. Additionally, we performed a genome-wide screen for genes that modulate the rate of ITS-induced GCRs. We find that mutation of core components of the DNA replication machinery leads to an increase in GCRs, but many mutants known to increase GCR rate in the absence of an ITS do not significantly affect GCR rate when an ITS is present. We also identified genes that promote the formation of ITS-induced GCRs, including genes with roles in telomere maintenance, nucleotide excision repair, and transcription. Our work thus uncovers multiple mechanisms by which an ITS promotes GCR. Significance statement Telomeric DNA repeats are found at the ends of linear chromosomes where they, together with specialized proteins that bind to them, protect chromosome ends from degradation and unwanted DNA repair activities. Telomeric repeats can also be found at internal locations in the genome, where they are called interstitial telomeric sequences (ITSs). ITSs are prone to breakage and are associated with human diseases. In this study, using baker’s yeast as a model organism, we show that instability at ITSs is driven by multiple factors, and identify genes that either promote or suppress gross chromosomal rearrangements induced by the presence of an ITS. Introduction Telomeres consist of repetitive DNA sequences and associated proteins, and are essential structures that ‘cap’ and protect the ends of eukaryotic chromosomes ( 1 ). A dysfunctional telomere will cause the chromosome end to be recognized as DNA damage, activating the DNA damage response and inhibiting cell proliferation. Telomeres, however, are also problematic because they are difficult to replicate. First, due to the enzymatic characteristics of DNA polymerases, the standard DNA replication machinery cannot fully replicate DNA ends—a problem famously referred to as the end replication problem ( 2 ). This problem is solved by an enzyme called telomerase, which adds telomeric repeats to the ends of the chromosomes ( 3 ). A second (and less-appreciated) problem is that DNA replication forks often stall and collapse while traversing telomeric sequences. This is highly conserved throughout evolution, having been reported in budding yeast Saccharomyces cerevisiae , fission yeast Schizosaccharomyces pombe , and mouse and human cells ( 4 – 7 ). There are at least three obstacles that could hinder the progression of the replication fork through telomeric sequences: 1) proteins that are tightly bound to telomeric sequences; 2) secondary structures (e.g. G-quadruplexes) that form due to the repetitive G-rich nature of telomeres ( 8 ); and 3) RNA-DNA hybrids formed from the transcription of telomeric repeat-containing RNA (TERRA; 9). If such impediments to replication fork progression are not properly dealt with, telomeres can become truncated and dysfunctional. The difficulty of replicating telomeric repeats extends to telomeric sequences located at internal regions of the genome, called interstitial telomeric sequences (ITSs). ITSs are naturally found in the genomes of many species and are thought to be either remnants of ancestral chromosomal fusions or erroneous insertions of telomeric repeats that occur during the repair of DNA double-strand breaks (DSBs; 10, 11). The human genome contains over 80 ITSs that have at least four telomeric repeats ( 12 ). ITSs are especially hazardous to genome stability because they are prone to breakage, provoke genome rearrangements, and are often found at translocation breakpoints in cancer cells ( 11 , 13 , 14 ). A break at an ITS can also be ‘healed’ by telomerase, resulting in the generation of a new telomere and loss of genetic material distal to the ITS. Such de novo telomere addition events have been linked to human disorders, including terminal deletions of chromosome 16 (alpha-thalassemia; 15, 16) and chromosome 12 (mental retardation; 17). In this study, we quantitatively measured ITS stability in S. cerevisiae using a gross chromosomal rearrangement (GCR) assay. We find that GCR rates rise exponentially with increasing ITS length. This increase is caused by the presence of the telomere repeat binding protein Rap1, which is known to impede DNA replication ( 5 , 18 – 20 ), and a strong preference for repairing DNA breaks at or distal to the ITS by de novo telomere addition. In addition, we performed a genome-wide screen to identify genes that contribute to ITS stability. We find that mutations in DNA replication genes elevate GCR rates, but many genes reported as general GCR suppressors do not exhibit an effect in the presence of an ITS. We also identified genes that promote ITS-induced GCRs, including genes important for telomere maintenance, nucleotide excision repair, and transcription. Our work reveals the multiple mechanisms by which an ITS promotes GCR. Results GCR rate increases exponentially with ITS length To quantitatively assess ITS stability, we modified the GCR assay developed by Chen and Kolodner by inserting an ITS of varying length between the most telomere-proximal essential gene on the left arm of chromosome V ( PCM1 ) and two counterselectable markers ( CAN1 and URA3 ) used in the original assay (21; Figure 1A ). The GCR rate (GCR events per cell division) can be determined by measuring the rate of simultaneous loss of CAN1 and URA3 —detected by growth on canavanine and 5-fluoroorotic acid (5-FOA), respectively— with a fluctuation test. We observe that the GCR rate increases with ITS length in two distinct exponential phases ( Figure 1B ; Table S1 ). Up to 50 bp, the GCR rate increases rapidly (1.6×10 -9 with no ITS to 4.5×10 -6 with a 50-bp ITS); beyond 50 bp, the GCR rate still increases exponentially, but more modestly (to 1.5×10 -4 with a 300-bp ITS). To rule out the possibility that the increase in GCR rate is simply due to the high G/C-content of the ITS, we inserted 300 bp of lambda phage DNA, which has a 62% G/C content, similar to wild-type telomeric sequence. The GCR rate of this strain is similar to that of the ‘no-ITS’ control ( Figure 1C ; Table S1 ). Telomeric sequences promote transcriptional silencing of adjacent genes ( 22 ). To determine whether the increase in GCR rate was in part caused by silencing of the CAN1 and URA3 genes, we measured the GCR rate in a 300-bp ITS-containing strain deleted for SIR2 , a gene needed for silencing ( 23 ). We find that absence of Sir2 does not affect the GCR rate ( Figure 1C ; Table S1 ). Thus, the ITS-induced increase in GCR rate is due neither to the high G/C-content of the ITS nor to silencing of the GCR markers. View this table: View inline View popup Download powerpoint Table S1. GCR rate increases with ITS length. Download figure Open in new tab Figure 1. GCR rate increases with ITS length due to Rap1-dependent inhibition of replication and a bias of repairing DNA breaks via de novo telomere addition. ( A ) Schematic diagram of the ITS-GCR assay. Loss of both URA3 and CAN1 allows growth on 5-FOA and canavanine. ITSs of varying length were inserted between CAN1 and PCM1 , the most telomere-proximal essential gene on the left arm of chromosome V. ( B ) GCR rate plotted as a function of ITS length. Wild-type ITS in black; tlc1-tm ITS in red. ( C ) GCR rates of strains with the indicated sequence insertions are plotted. Error bars represent SEM (n = 3 or 4). Two main factors can explain why GCR rate increases with ITS length. First, telomeric DNA is difficult to replicate, so the longer the ITS, the greater the possibility for a GCR event to occur. Second, if a DNA break occurs at or distal to the ITS, the probability that the break is ‘repaired’ by de novo telomere addition (resulting in a GCR) is very high if at least 34 bp of telomeric sequence is present on the centromere-side of the break. A sharp threshold separates a DSB from a telomere; DNA ends with 34 bp of telomeric sequence are recognized as critically short telomeres and efficiently extended by telomerase, whereas those with less than 34 bp are not ( 24 ). The bias towards repair via de novo telomere addition reaches a maximum once the ITS is sufficiently longer than this threshold; once a DNA end looks like a telomere, it cannot look even more like a telomere—at least in terms of de novo telomere addition. Thus, the increase in GCR rate is more modest once the ITS is longer than 50 bp. Note that the inflection point does not occur at 34 bp because a break could occur either within the ITS or distal to the ITS. For a 34-bp ITS, the break that leads to the de novo telomere addition most likely occurs distal to the ITS; if it were to occur within the ITS, the resulting DNA end would have less than 34 bp of telomeric sequence and would therefore not be efficiently extended by telomerase. For increasingly longer ITSs, the probability that the break occurs within the ITS increases, as does the probability of that break leaving at least 34 bp of telomeric sequence on the centromere-side of the break. Both of these probabilities will eventually reach a maximum (i.e. at a certain ITS length, almost all breaks will occur within the ITS, and almost all of these breaks will leave at least 34 bp of telomeric sequence on the centromere-side of the break), and further increases in ITS length will not affect these factors (although GCR rate will continue to increase due to Rap1-mediated fork collapse; see below). We do not know the exact ITS length where this effect occurs, but the net effect is an inflection point in the GCR rate curve at approximately 50 bp in ITS length. Rap1 promotes the ITS-induced increase in GCR rate The difficulty in replicating telomeric DNA has been attributed to the binding of Rap1 to telomeric repeats ( 5 , 18 – 20 ). To test whether Rap1 is promoting the ITS-induced increase in GCR rate, we examined the effect of an ITS with tlc1-tm [(TG) 0-4 TGG] n ATTTGG telomeric repeats ( 25 ), rather than wild-type (TG) 0-6 TGGGTGTG(G) 0-1 repeats ( 26 ). The tlc1-tm mutant repeats disrupt Rap1 association, but can still be efficiently recognized by telomerase and cap telomeres when located at chromosome ends ( 27 ). We find that the tlc1-tm ITS also increases GCR rate, but to a lesser extent than the wild-type ITS ( Figure 1B ; Table S1 ). The relationship between GCR rate and tlc1-tm ITS length is consistent with the DSB-telomere threshold, which is not affected by Rap1 binding ( 24 ), playing a dominant role. As tlc1-tm ITS length increases, repair by de novo telomere addition increases, reaching a maximum once the tlc1-tm ITS is sufficiently longer than the DSB-telomere threshold. However, the more modest exponential increase in GCR rate observed with wild-type ITS lengths above 50 bp is not seen with the tlc1-tm ITS. Since Rap1 is not completely depleted from tlc1-tm sequences ( 27 ), we also measured the effect of a 50-bp insertion of TG dinucleotides, i.e. (TG) 25 , which is predicted to disrupt Rap1 binding even more so than tlc1-tm repeats ( 28 ), but like tlc1-tm , can still be efficiently extended by telomerase ( 24 ). We find that the (TG) 25 -induced GCR rate is comparable to the 50-bp tlc1-tm ITS-induced GCR rate; i.e. there is an increase in the GCR rate, but much less than with a 50-bp wild-type ITS ( Figure 1C ; Table S2 ). The difficulty in replicating telomeric sequences has also been attributed to the formation of G-quadruplexes, which is compromised in TG dinucleotide and tlc1-tm repeats ( 29 ). Thus, we also tested 50 bp of TTGGGG Tetrahymena telomeric repeats, which can be extended by S. cerevisiae telomerase ( 30 ) and have the ability to form G-quadruplexes ( 31 ), but does not bind Rap1 ( 32 ). We find that this sequence increases GCR rate to a level slightly less than that of the 50-bp tlc1-tm ITS ( Figure 1C ; Table S2 ). These findings suggest that Rap1, rather than G-quadruplex formation, promotes the wild-type ITS-induced increase in GCR rate. View this table: View inline View popup Download powerpoint Table S2. GCR rates of strains with different 50-bp insertions. To further assess the ability of G-quadruplexes to induce GCR, we measured the effect of inserting 50 bp of the hexanucleotide repeats GGGGCC/GGCCCC and CCCTCT/AGAGGG on GCR rate. Expansion of these repeats cause neurogenerative diseases (amyotrophic lateral sclerosis and frontotemporal degeneration for GGGGCC/GGCCCC and X-linked dystonia parkinsonism for CCCTCT/AGAGGG), and the formation of G-quadruplexes by these repeats has been proposed to be involved in their expansion ( 33 ). The insertion of these sequences only mildly increases GCR rate ( Figure 1C ; Table S2 ). Our findings indicate that G-quadruplexes play a relatively minor role, in comparison to Rap1, in hindering the replication of S. cerevisiae telomeric DNA, in agreement with previous work, including an in vitro study examining the replication of telomeric sequences with and without Rap1 ( 5 , 20 ). Breaks occur within and distal to the ITS Since most GCR events detected in this assay without an ITS are predominantly de novo telomere addition events ( 34 ), we reasoned that the addition of an ITS would only further increase this bias, most likely with the new telomere added at the ITS. Thus, to gain insight into the mechanisms that underlie the increased GCR rates induced by the presence of an ITS, we sequenced the ITS region from independently isolated canavanine- and 5-FOA-resistant mutants (i.e. GCR survivors) derived from strains that either had the 50-bp ITS or the 100-bp ITS, 20 from each strain. As we expected, de novo telomere addition occurred at the ITS in all 40 GCR survivors. Since yeast telomerase adds imperfect, degenerate repeats ( 26 ), we can determine the exact position were telomerase added the new telomere by comparing with the original 50-bp and 100-bp ITS sequences ( Figure 2A ). Only a centromere-proximal portion of the ITS was retained in some of the GCR survivors (nine of 20 derived from the 50-bp ITS strain, 18 of 20 derived from the 100-bp ITS strain), suggesting that the DNA replication fork collapsed while traversing the ITS, followed by the telomerase-mediated addition of a new telomere. In all GCR survivors, at least 30 bp of the original ITS sequence was retained, consistent with DNA ends with less than 30 bp of telomeric sequence being recognized as DSBs and not efficiently extended by telomerase ( 24 ). Download figure Open in new tab Figure 2. Breaks occur within and distal to the ITS. ( A ) GCR survivors were isolated by growing strains with either a 50-bp ITS or 100-bp ITS on agar plates containing canavanine and 5-FOA. Twenty independent isolates from each strain were analyzed. In all 40 isolates, the presence of a de novo telomere added at the ITS site was confirmed by PCR and sequencing. The length of the original ITS retained for each de novo telomere is plotted. Four isolates also retained sequence downstream of the ITS and are indicated by an asterisk. ( B ) The sequences of the four isolates are aligned to DNA sequence from the original 50-bp ITS-containing strain (50.ref). The last 20 bp of the ITS are underlined. Newly added telomeric sequence in the GCR survivors is in bold and red. Surprisingly, the entire ITS sequence was retained in the remaining GCR survivors (11 of 20 derived from the 50-bp ITS strain, two of 20 derived from the 100-bp ITS strain). These findings suggest that the DNA replication fork likely collapsed distal to the ITS, followed by resection back to the ITS prior to telomere addition, leaving the original ITS intact. Consistent with our observations, it has been previously shown that sites of repair-associated telomere addition (SiRTAs; hot spots in the genome for de novo telomere addition) can be located several kilobases away from an induced DSB ( 35 ). In four GCR survivors derived from the 50-bp ITS strain, up to 10 bp immediately downstream of the ITS was also retained ( Figure 2B ). This is analogous to SiRTAs, which have a bipartite structure consisting of a Stim sequence and a Core sequence; Cdc13 can bind to a Stim sequence (in this case, the ITS) and stimulate de novo telomere addition at the downstream Core sequence ( 35 ). Identification of genes that suppress ITS-induced GCR events To identify genes that suppress ITS instability, we used a high-throughput replica-pinning approach that we recently developed to detect low-frequency events ( 36 ), such as GCRs. First, we introduced the 50-bp ITS GCR assay components into the yeast knockout (YKO) and conditional temperature-sensitive (ts) strain libraries ( 37 , 38 ) using the synthetic genetic array (SGA) method ( 39 ). The resulting strains were amplified by high-throughput replica-pinning, yielding 24 colonies per YKO or ts strain, onto media containing canavanine and 5-FOA to select for GCR events (see Materials and Methods for details). GCR frequencies (percentage of GCR-positive colonies) were calculated for each mutant strain ( Figure 3A ). Download figure Open in new tab Figure 3. Identification of genes that suppress ITS-induced GCR rate. ( A ) A high-throughput screen was performed as described in the text. All 24 replica-pinned colonies on media containing both canavanine and 5-FOA of the his3Δ control strain and three selected mutants with increased GCR frequencies are shown. ( B ) Putative hits from the high-throughput screen were tested in a patch-and-replica-plate assay. An example plate, with three mutants that tested positive (red boxes), is shown. A negative control ( his3Δ ) and a positive control ( ydl162cΔ ) were included on each plate. ( C ) Fold change in GCR rate of mutants identified in the ITS-GCR screen. ( D ) Fold change in GCR rate of ten known GCR suppressors not identified in the ITS-GCR screen. Fold change of mutants without an ITS is relative to a wild-type strain without an ITS. Fold change of mutants with a 50-bp ITS is relative to a wild-type strain with an ITS. Data for YKO strains without an ITS were taken from a previous publication ( 50 ). The median GCR frequency was 38% for the YKO strains and 42% for the ts strains. Only mutants with a GCR frequency above 70% (corresponding to a Fisher’s exact test P-value of <0.002) were analyzed further (Dataset S1). A total of 54 YKO and 44 ts mutants met this criterion. These strains were generated again by SGA and further tested using a patch-and-replica-plate approach ( Figure 3B ; 40), from which 21 mutants (5 YKO and 16 ts) appeared to have an increased ITS-induced GCR rate. Their GCR rates were determined by fluctuation tests. Ten of the 21 mutants have more than a twofold increase in ITS-induced GCR rate ( Figure 3C ; Table S3 ). View this table: View inline View popup Download powerpoint Table S3. GCR rates of mutants identified in the ITS-GCR screen without and with a 50-bp ITS. Of these nine genes (two of the mutants are different alleles of CDC2 ), eight are primarily involved in DNA replication ( CDC2 / POL3 , CDC9 , ELG1 , RAD27 , RFC2 , RFC4 , RFC5 , and YDL162c ). Pol3 is the catalytic subunit of DNA polymerase δ ( 41 , 42 ). Rfc2, Rfc4, and Rfc5 are small subunits of the replication factor C (RFC) complex, which catalyzes the loading of the proliferating cell nuclear antigen (PCNA) sliding clamp ( 43 ). Elg1 forms an alternative RFC complex with Rfc2–5 that is implicated in unloading PCNA ( 44 ). Rad27 is a flap endonuclease involved in Okazaki fragment processing and maturation ( 45 ). Cdc9 is DNA ligase I, responsible for ligating Okazaki fragments together ( 46 ). YDL162c is an open reading frame that overlaps with the CDC9 promoter; its deletion reduces CDC9 expression ( 47 ). The ninth gene, RPT4 , encodes an ATPase of the 19S regulatory particle of the 26S proteasome ( 48 ), which is important for many cellular functions, including DNA replication and genome stability ( 49 ). With the exception of RPT4 , all of these genes also suppress GCR rate in the absence of an ITS ( Figure 3C ; Table S3 ), which is not surprising given that these genes are involved in DNA replication; defects in DNA replication should increase GCRs regardless of whether an ITS is present. The ITS-specific increase in GCR rate observed in the rpt4-150 strain may indicate that the proteasome removes a GCR-inducing protein that is bound to the ITS. An obvious candidate for such a protein is Rap1, although we do not detect a change in cellular Rap1 levels in rpt4-150 cells ( Figure S1 ). However, other explanations for the ITS-specific effect of rpt4-150 are possible, and further studies are necessary to elucidate the mechanism. Download figure Open in new tab Figure S1. Rap1 protein levels in wild-type and rpt4-150 strains. Wild-type and rpt4-150 cells, harvested from logarithmically growing cultures, were fixed using TCA. Extracts from each sample were subjected to SDS-PAGE separation, followed by immunoblotting to detect Rap1 protein. Stain-Free imaging was used to assess the total protein input. Many mutants increase GCR rate in the absence of an ITS but not in its presence Many mutants have been reported to increase GCR rate in the absence of an ITS ( 50 , 51 ). Interestingly, the majority of these mutants were not identified in our screen and might be false negatives. Therefore, we measured the GCR rate of 10 of these mutants in the presence of a 50-bp ITS ( Figure 3D ; Table S4 ). Seven of these mutants do not significantly alter ITS-induced GCR rate (i.e. less than 1.7-fold; rad50Δ , sic1Δ , rlf2Δ , rmi1Δ , mus81Δ , slx8Δ, sgs1Δ ). For the remaining three, pif1Δ , rad52Δ , and slx5Δ increase ITS-induced GCR rate by 2.5-, 12-, and 2.7-fold, respectively, far lower than their effect on GCR rate in the absence of an ITS. Thus, it appears that many known GCR suppressors have a greatly reduced, if not completely absent, role in suppressing GCRs when an ITS is present. We also tested rrm3Δ because Rrm3 is a helicase that promotes replication fork progression through telomeric sequences ( 4 ). Deletion of RRM3 has been reported to exhibit a mild fourfold increase in GCR rate in the absence of an ITS ( 52 ); we find that this is also true in the presence of an ITS ( Table S4 ). View this table: View inline View popup Download powerpoint Table S4. GCR rates of selected mutants not identified in the ITS-GCR screen without and with a 50-bp ITS. Most known GCR suppressors have roles in DNA replication or repair. However, one exception that stands out is the cyclin-dependent kinase inhibitor Sic1. Cells lacking Sic1 initiate DNA replication from fewer origins of replication, causing an increase in the distance a replication fork has to travel and in the time needed to replicate the entire genome ( 53 ), including the region of chromosome V relevant for the GCR assay ( 54 ). It was suggested that ongoing replication as sic1Δ cells enter anaphase may lead to an increase in DSBs and GCRs ( 53 ). ITS-induced pausing of the replication fork would also cause a delay in replicating DNA distal to the ITS, resulting in under-replication and DNA breaks distal to the ITS, consistent with the data we obtained by sequencing GCR survivors ( Figure 2 ). The lack of an increase in GCR rate in sic1Δ strains with a 50-bp ITS suggests that the ITS is epistatic to sic1Δ in terms of delaying replication of the left arm of chromosome V. In other words, replication of the chromosomal region distal to the ITS is chronically delayed, so much so that further deletion of SIC1 cannot make it worse. The rfc2-1 and rfc5-1 mutations dramatically accelerate senescence in the absence of telomerase Mutations that increase ITS-induced GCR rate likely increase the probability of replication fork collapse within the ITS. Such mutations may also increase fork collapse at native telomeres, which could lead to truncation of telomeres and accelerated senescence in the absence of telomerase. Consistent with this idea, cdc2-2 and rad27Δ have both been reported to cause accelerated senescence in the absence of telomerase ( 55 , 56 ). We tested the remaining eight mutations that increase ITS-induced GCR rate. We sporulated diploids that are heterozygous in one of the genes of interest as well as EST2 , which encodes the protein catalytic subunit of telomerase ( 57 ), and followed the growth of the haploid meiotic progeny by serial propagation in liquid cultures for several days ( Figure 4 ). As expected, est2Δ cultures grew slower as the experiment progressed and cells senesced, but growth was eventually restored upon the emergence of survivors that utilize recombination-mediated mechanisms to maintain telomeres ( 58 ). We find that est2Δ rfc2-1 and est2Δ rfc5-1 double mutants senesce much faster than est2Δ single mutants. The cdc2-7 , cdc9-1 , and elg1Δ mutations appear to modestly accelerate senescence, while the rpt4-150 mutation causes a slight delay in senescence. Thus, seven of the 10 mutants found to elevate ITS-induced GCR rate also cause accelerated senescence in the absence of telomerase. Download figure Open in new tab Figure 4. The cdc2-7 , cdc9-1 , elg1Δ , rfc2-1 , and rfc5-1 mutations accelerate senescence in the absence of telomerase. Senescence was monitored by serial passaging of haploid meiotic progeny derived from the sporulation of ZYY238 ( A ), ZYY236 ( B ), ZYY232 ( C ), ZYY244 ( D ), ZYY246 ( E ), ZYY248 ( F ), ZYY240 ( G ), and ZYY228 ( H ). Average cell density ±SEM of three independent isolates per genotype is plotted. Identification of mutants that decrease ITS-induced GCR rate We repeated the genome-wide screen with minor adjustments that allowed us to identify mutants that decrease ITS-induced GCR rate (see Materials and Methods for details). The median GCR frequency was 79% for the YKO strains and 67% for the ts strains. Only mutants with a GCR frequency below 38% for the YKO strains and 31% for the ts strains (corresponding to a Fisher’s exact test P-value of <10 -4 ) were analyzed further ( Figure 5A ; Dataset S2). A total of 248 YKO and 114 ts mutants met this criterion. These strains were generated again by SGA and further tested using the patch-and-replica-plate approach ( Figure 5B ). A large number of the remaining genes have roles in chromosome segregation, spindle assembly checkpoint, and microtubule-based processes. The identification of these genes is due to the CIN8 gene located distal to the ITS on chromosome V (Yao and Yin et al., accompanying manuscript). De novo telomere addition at the ITS results in the loss of CIN8 , which is synthetic lethal with mutation of these genes. Thus, we eliminated the hits that are known to be synthetic lethal with cin8Δ . We also re-tested these mutants with the patch- and-replicate-plate approach after introducing an extra copy of CIN8 located elsewhere in the genome, and eliminated hits that did not show a decrease in ITS-induced GCRs. Finally, we performed an SGA analysis of the hits, identifying and eliminating those that showed a negative synthetic genetic interaction with truncation of chromosome V at the ITS. The remaining 180 (122 YKO and 58 ts) hits show a high degree of enrichment for genes involved in nucleotide excision repair and transcription ( Figure 5C ; Table S5 ). A subset of the mutants was subjected to fluctuation tests to determine their ITS-induced GCR rates ( Figure 5D ; Tables S5 and S6 ). View this table: View inline View popup Table S5. List of mutants identified in the screen that decrease ITS-induced GCRs. View this table: View inline View popup Download powerpoint Table S6. GCR rates of telomere and NER mutants with a 50-bp ITS. Download figure Open in new tab Figure 5. Identification of genes that promote ITS-induced GCR rate. ( A ) A high-throughput screen was performed as described in the text. All 24 replica-pinned colonies on media containing both canavanine and 5-FOA of the his3Δ control strain and three selected mutants with decreased GCR frequencies are shown. ( B ) Putative hits from the high-throughput screen were tested in a patch-and-replica-plate assay. An example plate, with ten mutants that tested positive (red boxes), is shown. A negative control ( his3Δ ) and a positive control ( bub2Δ ) were included on each plate. ( C ) The statistically supported GO terms enriched in the genes that promote ITS-induced GCR rate are shown. ( D ) GCR rates of the indicated strains containing a 50-bp ITS are plotted. Error bars represent SEM (n = 3–6). As expected, our screen identified EST1 , EST2 , and EST3 , which encode the three protein subunits of telomerase ( 59 ). Deleting either EST1 , EST2 , or EST3 has no significant effect on GCR rate in the absence of an ITS ( 60 ). In contrast, in the presence of an ITS, deletion of EST1 , EST2 , or EST3 decreases GCR rate by approximately 300-fold. However, est1Δ , est2Δ , and est3Δ strains with a 50-bp ITS have GCR rates that are still fivefold higher than a wild-type strain without an ITS, indicating that a 50-bp ITS can induce the formation of GCRs that are not the result of telomerase-mediated de novo telomere addition. These GCRs are still likely triggered by Rap1-mediated inhibition of replication, causing breaks either within or distal to the ITS that are repaired in a manner that leads to other types of GCRs, such as nonreciprocal translocations or interstitial deletions. The cdc13-1 and rap1-1 mutants exhibit 5.5- and 159-fold decreases, respectively, in GCR rate in the presence of a 50-bp ITS. The rap1-1 allele encodes a Rap1 mutant protein that is compromised in binding DNA ( 61 ), and may therefore be less capable of obstructing DNA replication and inducing GCR. The cdc13-1 result is consistent with our previous observation that this mutant is defective in generating a new telomere at a DNA end with 34 bp of telomeric sequence ( 24 ). Cdc13 binds to single-stranded telomeric DNA and is important for preventing excessive resection of telomere ends and for telomerase recruitment ( 62 , 63 ). The cdc13-1 allele is defective in the former function ( 62 ). It has been reported that defective resection promotes de novo telomere addition ( 64 ), so it is possible that excessive resection in the cdc13-1 mutant impairs de novo telomere addition. We also identified many genes ( RAD1 , RAD2 , RAD4 , RAD10 , RAD14 , RAD23 , RAD33 , and TFB5 ) required for nucleotide excision repair (NER). Deletion of RAD1 and RAD10 has previously been reported to reduce GCR formation in mutant strains with elevated GCR rates ( 65 ). Rad1 and Rad10 form a structure-dependent endonuclease that cleaves 3’ flaps at junctions between single- and double-strand DNA during NER as well as homology-dependent repair processes ( 66 ). It was proposed that Rad1-Rad10 may cleave 3’ flaps during the formation of a GCR, including the addition of a de novo telomere where the initiating break occurs distal to the addition site (65; Figure S2A ). If this model is correct, the fraction of GCR survivors that retain the entire ITS should decrease in rad1Δ or rad10Δ mutants. However, we find that this is not the case ( Figure S2B ), arguing against this model. Furthermore, the identification of many other genes involved in NER, which are not necessary for cleavage of 3’ flap structures, suggests instead that Rad1-Rad10 promote GCR formation within the context of the NER pathway. Download figure Open in new tab Figure S2. Deletion of RAD10 does not eliminate GCR survivors that retain the entire ITS. ( A ) Model for how the Rad1-Rad10 endonuclease could function during de novo telomere addition. A break that occurs distal to the ITS will undergo 5’–3’ end resection until a portion of the ITS becomes single stranded, which will then bind Cdc13 (not shown). Cdc13 recruits telomerase, followed by Rad1-Rad10 cleavage of the 3’ overhang/flap, allowing telomerase to extend the ITS into a de novo telomere. Deletion of RAD1 or RAD10 should reduce or eliminate GCR survivors that retain the entire ITS in the de novo telomere. ( B ) GCR survivors were isolated by growing rad10Δ strain with a 50-bp ITS on agar plates containing canavanine and 5-FOA. Nineteen independent isolates were analyzed. In all isolates, the presence of a de novo telomere added at the ITS site was confirmed by PCR and sequencing. The length of the original ITS retained for each de novo telomere is plotted. Nine out of 19 retain the entire ITS, including three isolates that also retained up to 14 bp of sequence downstream of the ITS (indicated by asterisks), contrary to what the model predicts. NER can be divided into two subpathways: global-genome NER (GG-NER) and transcription-coupled NER (TC-NER). The NER genes identified in the screen are important for both subpathways. In GG-NER, Rad7 and Rad16 form a stable complex that is needed for transcription-independent recognition of UV-induced damage, while Rad26 is involved in TC-NER ( 67 ). Deletion of RAD7 or RAD16 has no effect on GCR rate, while deletion of RAD26 decreases GCR rate approximately threefold ( Table S6 ), suggesting the involvement of TC-NER. It has been reported that R-loops, consisting of an RNA-DNA hybrid and displaced single-stranded DNA that can arise during transcription, are processed into DSBs by TC-NER ( 68 ). The identification of many genes with functions in transcription in our screen is consistent with R-loops promoting GCR via TC-NER. Regardless of the precise mechanism, the role of TC-NER in promoting GCR is independent of the ITS, because deletion of RAD1 or RAD10 also reduces GCR formation in strains without an ITS present ( 65 ). Discussion In this study, we examined ITS stability using a GCR assay in S. cerevisiae . We find that GCR rate increases exponentially with ITS length, which can be attributed to Rap1-mediated inhibition of replication of telomeric sequence, and to a bias in repairing DNA breaks by de novo telomere addition. The latter effect maximizes once the ITS is approximately 50 bp in length, as the ITS becomes longer than the DSB-telomere transition length of 34 bp. In addition, we performed a genome-wide screen and identified nine genes that contribute to the stability of telomeric sequences, most of whom encode core components of the DNA replication machinery. Surprisingly, many genes previously reported to suppress GCRs in the absence of an ITS do not do so in the presence of an ITS, highlighting the ability of an ITS to override mechanisms that normally suppress GCRs. We also find that genes with roles in telomere maintenance, NER, and transcription can facilitate the formation of ITS-induced GCRs. Binding of Rap1 to an ITS promotes its instability Our findings confirm and extend previous studies showing that DNA-bound Rap1, much more so than G-quadruplex formation, hinders replication fork progression through telomeric sequences and induces DNA breakage ( 5 , 18 , 20 ). We show that these breaks can occur within the ITS, but also distal to the ITS. The breaks distal to the ITS are likely caused by incomplete replication of DNA distal to the ITS because of Rap1-mediated delay of fork progression at the ITS. Unexpectedly, we find that the GCR rate increases exponentially with ITS length ( Figure 1B ). This suggests that Rap1 molecules are acting in a cooperative manner to obstruct DNA replication. The Rap1-interacting proteins Rif1 and Rif2 connect neighboring and distal DNA-bound Rap1 units to form a high-order interlinked scaffold ( 69 ), the properties of which could change as telomere length increases. However, DNA-bound Rap1 obstructs replication independently of Rif1 and Rif2 ( 5 , 18 , 20 ). Rap1 has been reported to interact with DNA in multiple binding modes ( 70 ) and can, by itself, induce local DNA stiffening ( 71 ). Further studies are needed to determine whether these properties of Rap1 can explain why GCR rate increases exponentially with telomere length. Defects in DNA replication increase ITS-induced GCR rate and accelerate replicative senescence We identified nine genes that suppress ITS-induced GCR rate ( Figure 3C ). Most have a direct function during DNA replication. If these mutants increase GCR rate by increasing DNA breakage, they may also increase breaks at native telomeres that would accelerate senescence in the absence of telomerase. This is already known to be true for cdc2-2 and rad27Δ ( 55 , 56 ), and we show that cdc2-7 , cdc9-1 , elg1Δ , rfc2-1 , and rfc5-1 also do so, especially the latter two ( Figure 4 ). The dramatic effect of rfc2-1 and rfc5-1 on senescence is somewhat surprising given that they do not have the largest effects on GCR rate, with or without an ITS present, and the rfc4-20 mutant does not significantly affect senescence at all. However, analysis of the genetic interaction profile similarity subnetwork for the identified genes, extracted from the global network ( 72 ), reveals that rfc2-1 and rfc5-1 , while clustering among DNA replication and repair genes, are positioned further away than the other identified replication genes ( Figure S3 ), indicating related but distinct functions. Consistent with this idea, Rfc2 and Rfc5 are unique among the small RFC subunits in that they are about twice as abundant as Rfc3 and Rfc4 ( 73 ), and can form a stable heterodimer that acts on PCNA in vitro ( 74 ). rfc2-1 rfc5-1 double mutants are inviable, and overexpression of RFC5 can suppress the temperature sensitivity of rfc2-1 ( 75 ). Furthermore, the temperature sensitivity and replication defect of the rfc5-1 mutant can be suppressed by overexpression POL30 , which encodes PCNA ( 76 ), suggesting that there is a deficiency in PCNA loading in this mutant. Thus, insufficient loading of PCNA may lead to replication defects that induce GCRs as well as accelerated senescence in the absence of telomerase. Download figure Open in new tab Figure S3. Genetic interaction profile similarity subnetwork for genes that suppress ITS-induced GCR rate. The network was generated using TheCellMap.org ( 2 ). Nodes (representing deletions of nonessential genes or temperature-sensitive alleles of essential genes) sharing similar genetic interaction profiles (PCC > 0.2) are connected by an edge in the network. Genes sharing similar genetic interaction profiles map closer to each other. The subnetwork was annotated using Spatial Analysis of Functional Enrichment (SAFE; 3). It has been reported that the large subunit of RFC is aberrantly cleaved in Hutchinson-Gilford progeria syndrome (HGPS), resulting in defective loading of PCNA ( 77 ). HGPS is a disease characterized by accelerated aging and is caused by a mutation of the LMNA gene, leading to the expression of a truncated form of lamin A called progerin ( 78 ). At the cellular level, expression of progerin alters nuclear morphology and function, nucleolar activity, mitochondrial function, nucleocytoplasmic trafficking, and telomere homeostasis ( 79 ). It is still unclear how these alterations ultimately lead to the clinical features of HGPS, but telomere dysfunction may play a key role given that HGPS is associated with accelerated telomere erosion ( 80 ), and expression of telomerase improves the fitness of HGPS cells ( 81 – 83 ). Our findings support the hypothesis that premature senescence in HGPS is caused by a deficiency in PCNA loading and replication fork collapse at telomeres. Interestingly, many of the other DNA replication genes identified in our screen have roles in lagging strand synthesis and Okazaki fragment processing. Elg1, in complex with Rfc2–5, unloads PCNA following Okazaki fragment ligation by Cdc9; depletion of Elg1 or Cdc9 causes accumulation of PCNA on DNA, which leads to genome instability ( 44 , 84 – 87 ). Cells lacking Rad27 accumulate unligated Okazaki fragments ( 88 ), and presumably PCNA on DNA. Defects in lagging strand synthesis in the DNA polymerase δ mutants cdc2-2 and cdc2-7 would likely also increase the abundance of unligated Okazaki fragments and DNA-bound PCNA. Thus, a defect in unloading PCNA from DNA may also increase the probability of GCR. Presence of an ITS heavily biases repair towards de novo telomere addition Surprisingly, our screen identified neither PIF1 nor RRM3 , which encode Pif1-family helicases. Stalling of replication forks traversing telomeric sequences increases in the absence of Rrm3, which is important for replication past nonhistone protein-DNA complexes ( 4 , 89 ). Pif1 performs several functions that impact telomere biology, including inhibition of telomerase ( 90 , 91 ), Okazaki fragment processing ( 92 ), unwinding of G-quadruplexes ( 93 , 94 ), and promoting replication of Rap1-bound telomeric DNA ( 19 , 20 ). Other genes encoding proteins known to suppress GCR in the absence of an ITS were also not identified, such as the MRX (Mre11-Rad50-Xrs2) complex, the STR (Sgs1-Top3-Rmi1) complex, the Mus81 endonuclease, and the SUMO-targeted ubiquitin ligase Slx5-Slx8. As they may have been false negatives in our screen, we tested deletions of some of these genes directly in the ITS-GCR assay. Deletion of PIF1 , which has been reported to cause an approximately 1000-fold increase in GCR rate in the absence of an ITS ( 60 ), only led to a 2.5-fold increase in the presence of a 50-bp ITS ( Figure 3D ; Table S4 ). We speculate that the role of Pif1 in suppressing GCR is primarily due to its function in suppressing de novo telomere addition ( 90 ), a function that is negated if the DNA end contains more than 34 bp of telomeric sequence ( 24 ). The residual 2.5-fold increase in the presence of the ITS could be due to the role of Pif1 in overcoming Rap1-dependent inhibition of DNA replication ( 19 , 20 ), or its role in unwinding G-quadruplexes ( 93 , 94 ). Consistent with that latter hypothesis, it has been previously reported that the GCR rate of the pif1-m2 mutant, which is deficient in nuclear Pif1, is increased threefold in the presence of G-quadruplex motif sequences ( 94 ). Strains lacking any member of the MRX complex have an approximately 600-fold increase in GCR rate in the absence of an ITS ( 21 ). Deletion of SGS1 or TOP3 increases GCR rate by 20–30-fold, while deletion of RMI1 increases GCR rate by more than 170-fold ( 50 , 95 , 96 ). Deletion of MUS81 increases GCR rate 100–200-fold, and deletion of SLX5 or SLX8 increases GCR rate by more than 60-fold ( 50 , 65 , 97 ). We find that deletion of either RAD50 , SGS1 , RMI1 , MUS81 , SLX5 , or SLX8 has a small or negligible effect on GCR rate in the presence of a 50-bp ITS ( Figure 3D ; Table S4 ). The MRX and STR complexes are central players in the repair of DSBs ( 98 , 99 ); Mus81 is a structure-specific endonuclease that resolves replication and recombination intermediates ( 100 ); Slx5-Slx8 plays diverse roles in genome maintenance ( 101 ). In the absence of an ITS, disruption of these complexes will increase the likelihood of inappropriate repair that leads to a GCR. This effect is likely negated when the DNA break occurs at or distally adjacent to an ITS, since these breaks would be healed by de novo telomere addition regardless of whether these complexes are present. Consistent with this idea, a DSB that has telomeric repeats on one side of the DSB will result in asymmetric processing of the ends: the non-telomeric end will recruit the MRX complex and move to the nuclear pore complex while the telomeric side will not, favoring elongation by telomerase ( 102 ). In summary, we have shown that ITSs are unstable and promote the formation of GCRs via multiple mechanisms. First, Rap1 impedes ITS replication, which increases the probability of fork collapse within the ITS. Second, impeding replication at the ITS will also increase fork collapse distal to the ITS due to incomplete replication of downstream DNA. Third, a DSB within or distal to the ITS, whether caused by fork collapse or other means, will bias repair towards de novo telomere addition, especially if at least 34 bp of telomere sequence remains on one side of the DSB. Lastly, transcription and NER can facilitate the formation of ITS-induced GCRs. Some of these mechanisms are also at play at native telomeres. For example, Rap1 also impedes replication at native telomeres, and a DSB within a telomere will be preferentially healed by telomerase, but this is not a problem precisely because it is desirable for a truncated telomere to be extended by telomerase. Thus, mechanisms that have evolved for proper telomere function are sources of instability and GCR at an ITS. Materials and methods Yeast strains and media All yeast strains used in this study are listed in Table S7 . To prevent the onset of replicative senescence, the est strains used in Figure 5C were generated by replacing the corresponding EST gene with a kanMX cassette in ZYY141, and then used immediately for fluctuation tests without storing the strains. Standard yeast genetic and molecular methods were used ( 103 , 104 ). Experiments involving a strain containing a ts allele were performed at 22°C; in all other experiments, yeast strains were cultured at 30°C. ITS and other DNA sequences were inserted in GCR assay-containing strains at the PRB1 locus and are listed in Table S8 . View this table: View inline View popup Table S7. Yeast strains used in this study. View this table: View inline View popup Download powerpoint Table S8. ITS and other inserted DNA sequences Fluctuation tests of GCR rates Fluctuation tests for the quantification of GCR rates were performed essentially as previously described ( 105 ) by transferring entire single colonies from YPD plates to 4 ml of YPD liquid medium. Cultures were grown to saturation (30°C for YKO strains and 22°C for ts mutants). 50 µl of a 10 5 -fold dilution were plated in YPD plates. An strain-dependent quantity of cells was plated on SD-arg+canavanine+5-FOA. Colonies were counted after incubation at 30ᵒC or 22ᵒC for 3–7 days. The number of GCR (canavanine- and 5-FOA-resistant) colonies was used to calculate the GCR rate by the method of the median ( 106 ). PCR and sequencing of GCR survivors Genomic DNA from GCR survivors was isolated using a Wizard Genomic DNA Purification Kit (Promega). 1 µl of genomic DNA (∼100 ng) was mixed with 8 µl of 1x Cutsmart buffer (New England Biolabs (NEB), Ipswich, MA) and boiled for 10 min at 94ᵒC. 1 µl of tailing mix (0.05 µl Terminal Transferase (NEB, cat. no. M0315), 0.1 µl 10x Cutsmart buffer, 0.1 µl 10 mM dCTP, 0.75 µl dH 2 O) was added and incubated for 30 min at 37ᵒC, 10 min at 65ᵒC, and 5 min at 96ᵒC. Immediately after tailing, 30µl of PCR mix was added. The PCR mix consisted of 4 µl 10x PCR buffer (670 mM Tris-HCl pH 8.8, 160 mM (NH 4 ) 2 SO 4 , 50% glycerol, 0.1% Tween-20), 0.32 µl 25 mM dNTP mix, 0.3 µl 100 µM site-specific primer (5’-TTTTCGCCTCGACATCATCTGC-3’), 0.3 µL 100 µM G 18 primer (5’-CGGGATCCG 18 -3’), 0.5 µl Q5 High-Fidelity DNA Polymerase (NEB, cat. no. M0491), 24.68 µl dH 2 O. The samples were denatured at 98°C for 3 min, followed by 35 cycles of 98°C for 30 s, 68°C for 15 s, and a final extension step at 72°C for 2 min. PCR products were separated on 2.5% agarose gels and extracted using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany, cat. no. 740609). The purified PCR products were then cloned using a Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific, cat. no. 450245). Individual clones were sequenced by Eurofins Genomics (Germany) and the resulting data were analyzed using Sequencher software (Gene Codes, Ann Arbor, MI). Genome-wide screen for mutants with increased ITS-GCR rate The high-throughput replica-pinning screen was performed essentially as previously described ( 36 ) using a ROTOR-HDA pinning robot (Singer Instruments). In brief, prb1ΔhphMX-50bp_ITS and hxt13ΔURA3 were introduced into the YKO and ts libraries, with each strain of the libraries present in quadruplicate, by the SGA method ( 39 ). The screen was performed at 30°C and 22°C for the YKO and ts strains, respectively. Strains in which less than two colonies (out of a possible four) grew on the final SGA plates were excluded from further analysis. Each plate of the resulting prb1ΔhphMX-50bp_ITS hxt13ΔURA3 xxx::kanMX strains (where xxx::kanMX indicates either a gene knockout or ts allele) was then replica pinned twice in succession onto six SD-arginine+canavanine+5-FOA plates, yielding 24 colonies per strain. Only hits with a Fisher’s exact test P-value less than 0.002, comparing to the observed number of colonies on selective and non-selective plates for all strains in the screen, were selected for validation, which occurred in two steps. First, for only the screen hits, the prb1ΔhphMX-50bp_ITS hxt13ΔURA3 xxx::kanMX strains were generated again by the SGA method. Each strain was then streaked in a 1 cm x 1.5 cm patch on an SD-uracil plate, incubated at 30°C (for YKO strains) or 22°C (for ts strains) for 24-48 h, replica-plated on SD-arginine+canavanine+5-FOA to detect GCR events, and scored by visual inspection. Twenty-one strains (5 YKO and 16 ts) passed this validation step. These YKO or ts mutants were tested with one fluctuation test, and if an increase in ITS-GCR rate was observed, the mutants were introduced into a different strain background (W303), and then subsequently subjected to at least three additional fluctuation tests. Rap1 immunoblotting Cells were harvested and fixed using 20% trichloroacetic acid (TCA). A FastPrep 5G system (MP Biomedicals) was employed for cell disruption, following the recommended program for S. cerevisiae . An additional 5% TCA was added to adjust the final TCA concentration to 7.3%. The samples were then centrifuged at 14,000 rpm for 10 min at 4°C, after which the supernatant was discarded. Samples were resuspended in 1x loading buffer and loaded on 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked in 2.5% bovine serum albumin (BSA) for 60 min at room temperature and incubated with Rap1 antibody (sc-374297; Santa Cruz Biotechnology) in 2.5% BSA overnight at 4°C. The membranes were washed three times with 1× TBS containing 0.1% Tween 20 (Sigma) and incubated with m-IgGκ BP-HRP antibody (sc-516102; Santa Cruz Biotechnology) in 2.5% BSA for 2 h at room temperature. Blots were visualized using the Gel Doc XR+ System (Bio-Rad Laboratories). Liquid culture senescence assay Liquid culture senescence assays were performed essentially as previously described ( 107 ). Each senescence assay started with diploid strains. Freshly dissected haploid spores were allowed to form colonies on YPD agar plates after two days of growth at 30°C or 22°C. Cells from these colonies were serially passaged in liquid culture medium at 24-h intervals. For each passage, the cell density of each culture was measured by optical density (calibrated by cell counting using a haemocytometer), and the cultures were diluted back into fresh medium at a cell density of 2 × 10 5 cells/ml. Cell density was plotted as a function of population doublings. Genome-wide screen for mutants with decreased ITS-GCR rate To screen for mutants with decreased ITS-GCR rate, the high-throughput replica-pinning screen was repeated with a few modifications. In brief, the SGA-derived prb1ΔhphMX-50bp_ITS hxt13ΔURA3 xxx::kanMX strains were replica pinned onto six YPD+G418+hygromycin B, yielding 24 colonies per strain. The strains were then re-pinned twice in succession onto SD-arginine+canavanine+5-FOA plates. Only hits with a Fisher’s exact test P-value less than 10 -4 were selected for validation. A lower P-value was used due to the higher number of hits. Validation occurred in several steps. First, for only the screen hits, the prb1ΔhphMX-50bp_ITS hxt13ΔURA3 xxx::kanMX strains were generated again by the SGA method. Each strain was then streaked in a 1 cm x 1.5 cm patch on an SD-uracil plate, incubated at 30°C (for YKO strains) or 22°C (for ts strains) for 24-48 h, replica-plated on SD-arginine+canavanine+5-FOA to detect GCR events, and scored by visual inspection. Second, the strains were generated again by the SGA method, but with an extra copy of CIN8 ( ho::CIN8-natMX ) also introduced into the YKO and ts strains. These strains were also tested using the patch-and-replica-plate approach. Third, a GCR survivor (ZYY261) was generating by growing FRY910 (the SGA starting strain with the 50-bp ITS and GCR markers) on media containing canavanine and 5-FOA. ZYY261 was confirmed by PCR and sequencing to have a de novo telomere added at the ITS site. ZYY261 was crossed to the hits from the screen using the SGA method, and resulting strains that showed a synthetic genetic growth defect by visual comparison of colony sizes were eliminated from further analysis. Lastly, due to the large number of hits that passed the first three validation steps, only a subset of these was subsequently subjected to at least three fluctuation tests, and the mutants were not tested in a different strain background. Gene ontology enrichment analysis The GO term finder tool ( http://go.princeton.edu/ ) was used to query biological process enrichment for each gene set, with a P-value cutoff of 0.01 and Bonferroni correction applied. REVIGO ( 108 ) was used to further analyze the GO term enrichment data, using the “Medium (0.7)” term similarity filter and the simRel score as semantic similarity measure. As a result, terms with a frequency more than 10% in the REVIGO output were eliminated for being too broad. Competing Interests The authors declare no competing interests. Acknowledgments and funding sources We thank G.W. Brown for providing yeast strains; D. Novarina and L.M. Veenhoff for critical reading of the manuscript. F.R.R.B. was supported by a Consejo Nacional de Ciencia y Tecnología (CONACYT) scholarship. Z. Yin was supported by a scholarship from the Nanjing Huimou Medi-Tech Co. Y.Y. was supported by scholarships from the University of Groningen’s Abel Tasman Talent and from the Chongqing Explorer Decoration Engineering Co. Work in the laboratory of M. Chang was supported by an Open Competition M-2 grant from the Dutch Research Council. Footnotes Classifications: Biological Sciences (major), Cell Biology (minor) References 1. ↵ D. Jain , J. P. Cooper , Telomeric strategies: means to an end . Annu Rev Genet 44 , 243 – 269 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ R. J. Wellinger , In the end, what’s the problem? Mol Cell 53 , 855 – 856 ( 2014 ). OpenUrl PubMed 3. ↵ E. H. Blackburn , K. Collins , Telomerase: an RNP enzyme synthesizes DNA . Cold Spring Harb Perspect Biol 3 ( 2011 ). 4. ↵ A. S. Ivessa , J. Q. Zhou , V. P. Schulz , E. K. Monson , V. A. Zakian , Saccharomyces Rrm3p, a 5’ to 3’ DNA helicase that promotes replication fork progression through telomeric and subtelomeric DNA . Genes Dev 16 , 1383 – 1396 ( 2002 ). OpenUrl Abstract / FREE Full Text 5. ↵ S. Makovets , I. Herskowitz , E. H. Blackburn , Anatomy and dynamics of DNA replication fork movement in yeast telomeric regions . Mol Cell Biol 24 , 4019 – 4031 ( 2004 ). OpenUrl Abstract / FREE Full Text 6. K. M. Miller , O. Rog , J. P. Cooper , Semi-conservative DNA replication through telomeres requires Taz1 . Nature 440 , 824 – 828 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 7. ↵ A. Sfeir et al. , Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication . Cell 138 , 90 – 103 ( 2009 ). OpenUrl CrossRef PubMed Web of Science 8. ↵ M. L. Bochman , K. Paeschke , V. A. Zakian , DNA secondary structures: stability and function of G-quadruplex structures . Nat Rev Genet 13 , 770 – 780 ( 2012 ). OpenUrl CrossRef PubMed 9. J. Rivosecchi , K. Jurikova , E. Cusanelli , Telomere-specific regulation of TERRA and its impact on telomere stability . Semin Cell Dev Biol 157 , 3 – 23 ( 2024 ). OpenUrl 10. A. Ruiz-Herrera , S. G. Nergadze , M. Santagostino , E. Giulotto , Telomeric repeats far from the ends: mechanisms of origin and role in evolution . Cytogenet Genome Res 122 , 219 – 228 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 11. ↵ A. Y. Aksenova , S. M. Mirkin , At the Beginning of the End and in the Middle of the Beginning: Structure and Maintenance of Telomeric DNA Repeats and Interstitial Telomeric Sequences . Genes (Basel) 10 ( 2019 ). 12. ↵ S. G. Nergadze , M. A. Santagostino , A. Salzano , C. Mondello , E. Giulotto , Contribution of telomerase RNA retrotranscription to DNA double-strand break repair during mammalian genome evolution . Genome Biol 8 , R260 ( 2007 ). 13. ↵ A. Ruiz-Herrera et al. , Evolutionary breakpoints are co-localized with fragile sites and intrachromosomal telomeric sequences in primates . Cytogenet Genome Res 108 , 234 – 247 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 14. ↵ A. Y. Aksenova et al. , Genome rearrangements caused by interstitial telomeric sequences in yeast . Proc Natl Acad Sci U S A 110 , 19866 – 19871 ( 2013 ). OpenUrl Abstract / FREE Full Text 15. A. O. Wilkie , J. Lamb , P. C. Harris , R. D. Finney , D. R. Higgs , A truncated human chromosome 16 associated with α thalassaemia is stabilized by addition of telomeric repeat (TTAGGG) n . Nature 346 , 868 – 871 ( 1990 ). OpenUrl CrossRef PubMed Web of Science 16. J. Lamb et al. , De novo truncation of chromosome 16p and healing with (TTAGGG) n in the α-thalassemia/mental retardation syndrome (ATR-16) . Am J Hum Genet 52 , 668 – 676 ( 1993 ). OpenUrl PubMed Web of Science 17. A. C. Wong et al. , Molecular characterization of a 130-kb terminal microdeletion at 22q in a child with mild mental retardation . Am J Hum Genet 60 , 113 – 120 ( 1997 ). OpenUrl PubMed Web of Science 18. ↵ G. H. Goto et al. , Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication . PLoS Genet 11 , e1005283 ( 2015 ). OpenUrl CrossRef 19. ↵ K. N. Koc , S. P. Singh , J. L. Stodola , P. M. Burgers , R. Galletto , Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase delta . Nucleic Acids Res 44 , 3811 – 3819 ( 2016 ). OpenUrl CrossRef PubMed 20. ↵ M. E. Douglas , J. F. X. Diffley , Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro . Nucleic Acids Res 49 , 5671 – 5683 ( 2021 ). OpenUrl CrossRef 21. ↵ C. Chen , R. D. Kolodner , Gross chromosomal rearrangements in Saccharomyces cerevisiae replication and recombination defective mutants . Nat Genet 23 , 81 – 85 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 22. ↵ D. E. Gottschling , O. M. Aparicio , B. L. Billington , V. A. Zakian , Position effect at S. cerevisiae telomeres: Reversible repression of Pol II transcription . Cell 63 , 751 – 762 ( 1990 ). OpenUrl CrossRef PubMed Web of Science 23. ↵ O. M. Aparicio , B. L. Billington , D. E. Gottschling , Modifiers of position effect are shared between telomeric and silent mating-type loci in S. cerevisiae . Cell 66 , 1279 – 1287 ( 1991 ). OpenUrl CrossRef PubMed Web of Science 24. ↵ J. Strecker et al. , A sharp Pif1-dependent threshold separates DNA double-strand breaks from critically short telomeres . eLife 6 , e23783 ( 2017 ). OpenUrl CrossRef 25. ↵ M. Chang , M. Arneric , J. Lingner , Telomerase repeat addition processivity is increased at critically short telomeres in a Tel1-dependent manner in Saccharomyces cerevisiae . Genes Dev 21 , 2485 – 2494 ( 2007 ). OpenUrl Abstract / FREE Full Text 26. ↵ K. Förstemann , J. Lingner , Molecular basis for telomere repeat divergence in budding yeast . Mol Cell Biol 21 , 7277 – 7286 ( 2001 ). OpenUrl Abstract / FREE Full Text 27. ↵ F. R. Rosas Bringas , S. Stinus , P. de Zoeten , M. Cohn , M. Chang , Rif2 protects Rap1-depleted telomeres from MRX-mediated degradation in Saccharomyces cerevisiae . eLife 11 , e74090 ( 2022 ). OpenUrl 28. ↵ I. R. Graham , A. Chambers , Use of a selection technique to identify the diversity of binding sites for the yeast RAP1 transcription factor . Nucleic Acids Res 22 , 124 – 130 ( 1994 ). OpenUrl CrossRef PubMed Web of Science 29. ↵ S. Stinus , F. R. Rosas Bringas , L. Wanders , M. Chang , Investigating the role of G-quadruplexes at Saccharomyces cerevisiae telomeres . Microbial Cell 9 , 126 – 132 ( 2022 ). OpenUrl 30. ↵ J. Shampay , J. W. Szostak , E. H. Blackburn , DNA sequences of telomeres maintained in yeast . Nature 310 , 154 – 157 ( 1984 ). OpenUrl CrossRef PubMed Web of Science 31. ↵ J. R. Williamson , M. K. Raghuraman , T. R. Cech , Monovalent cation-induced structure of telomeric DNA: the G-quartet model . Cell 59 , 871 – 880 ( 1989 ). OpenUrl CrossRef PubMed Web of Science 32. ↵ J. Berman , C. Y. Tachibana , B. K. Tye , Identification of a telomere-binding activity from yeast . Proc Natl Acad Sci U S A 83 , 3713 – 3717 ( 1986 ). OpenUrl Abstract / FREE Full Text 33. ↵ A. N. Khristich , S. M. Mirkin , On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability . J Biol Chem 295 , 4134 – 4170 ( 2020 ). OpenUrl Abstract / FREE Full Text 34. ↵ K. Myung , A. Datta , R. D. Kolodner , Suppression of spontaneous chromosomal rearrangements by S phase checkpoint functions in Saccharomyces cerevisiae . Cell 104 , 397 – 408 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 35. ↵ U. C. Obodo et al. , Endogenous hotspots of de novo telomere addition in the yeast genome contain proximal enhancers that bind Cdc13 . Mol Cell Biol doi: 10.1128/mcb.00095-16 ( 2016 ). OpenUrl CrossRef 36. ↵ D. Novarina , F. R. Rosas Bringas , O. G. Rosas Bringas , M. Chang , High-throughput replica-pinning approach to screen for yeast genes controlling low-frequency events . STAR Protoc 3 , 101082 ( 2022 ). OpenUrl 37. ↵ G. Giaever et al. , Functional profiling of the Saccharomyces cerevisiae genome . Nature 418 , 387 – 391 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 38. ↵ Z. Li et al. , Systematic exploration of essential yeast gene function with temperature-sensitive mutants . Nat Biotechnol 29 , 361 – 367 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 39. ↵ E. Kuzmin , M. Costanzo , B. Andrews , C. Boone , Synthetic Genetic Array Analysis . Cold Spring Harb Protoc 2016 , pdb prot088807 (2016). 40. M. Chang , A. B. Parsons , B. H. Sheikh , C. Boone , G. W. Brown , Genomic approaches for identifying DNA damage response pathways in S. cerevisiae . Methods Enzymol 409 , 213 – 235 ( 2006 ). OpenUrl CrossRef PubMed 41. ↵ K. C. Sitney , M. E. Budd , J. L. Campbell , DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene . Cell 56 , 599 – 605 ( 1989 ). OpenUrl CrossRef PubMed Web of Science 42. ↵ A. Boulet , M. Simon , G. Faye , G. A. Bauer , P. M. Burgers , Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III . EMBO J 8 , 1849 – 1854 ( 1989 ). OpenUrl PubMed Web of Science 43. ↵ N. Y. Yao , M. O’Donnell , The RFC clamp loader: structure and function . Subcell Biochem 62 , 259 – 279 ( 2012 ). OpenUrl CrossRef PubMed 44. ↵ T. Kubota , K. Nishimura , M. T. Kanemaki , A. D. Donaldson , The Elg1 replication factor C-like complex functions in PCNA unloading during DNA replication . Mol Cell 50 , 273 – 280 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 45. ↵ Y. Liu , H. I. Kao , R. A. Bambara , Flap endonuclease 1: a central component of DNA metabolism . Annu Rev Biochem 73 , 589 – 615 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 46. ↵ L. H. Johnston , K. A. Nasmyth , Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase . Nature 274 , 891 – 893 ( 1978 ). OpenUrl CrossRef PubMed 47. ↵ X. Pan et al. , A DNA integrity network in the yeast Saccharomyces cerevisiae . Cell 124 , 1069 – 1081 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 48. ↵ H. B. McDonald , A. H. Helfant , E. M. Mahony , S. K. Khosla , L. Goetsch , Mutational analysis reveals a role for the C terminus of the proteasome subunit Rpt4p in spindle pole body duplication in Saccharomyces cerevisiae . Genetics 162 , 705 – 720 ( 2002 ). OpenUrl Abstract / FREE Full Text 49. ↵ J. J. Morgan , L. J. Crawford , The Ubiquitin Proteasome System in Genome Stability and Cancer . Cancers (Basel) 13 ( 2021 ). 50. ↵ C. D. Putnam et al. , Bioinformatic identification of genes suppressing genome instability . Proc Natl Acad Sci U S A 109 , E3251 – 3259 ( 2012 ). OpenUrl Abstract / FREE Full Text 51. ↵ C. D. Putnam , R. D. Kolodner , Pathways and Mechanisms that Prevent Genome Instability in Saccharomyces cerevisiae . Genetics 206 , 1187 – 1225 ( 2017 ). OpenUrl Abstract / FREE Full Text 52. ↵ K. H. Schmidt , R. D. Kolodner , Suppression of spontaneous genome rearrangements in yeast DNA helicase mutants . Proc Natl Acad Sci U S A 103 , 18196 – 18201 ( 2006 ). OpenUrl Abstract / FREE Full Text 53. ↵ A. Lengronne , E. Schwob , The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G1 . Mol Cell 9 , 1067 – 1078 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 54. ↵ P. Ayuda-Duran et al. , The CDK regulators Cdh1 and Sic1 promote efficient usage of DNA replication origins to prevent chromosomal instability at a chromosome arm . Nucleic Acids Res 42 , 7057 – 7068 ( 2014 ). OpenUrl CrossRef PubMed Web of Science 55. ↵ Y. L. Tsai , S. F. Tseng , S. H. Chang , C. C. Lin , S. C. Teng , Involvement of replicative polymerases, Tel1p, Mec1p, Cdc13p, and the Ku complex in telomere-telomere recombination . Mol Cell Biol 22 , 5679 – 5687 ( 2002 ). OpenUrl Abstract / FREE Full Text 56. ↵ J. Parenteau , R. J. Wellinger , Differential processing of leading- and lagging-strand ends at Saccharomyces cerevisiae telomeres revealed by the absence of Rad27p nuclease . Genetics 162 , 1583 – 1594 ( 2002 ). OpenUrl Abstract / FREE Full Text 57. ↵ J. Lingner et al. , Reverse transcriptase motifs in the catalytic subunit of telomerase . Science 276 , 561 – 567 ( 1997 ). OpenUrl Abstract / FREE Full Text 58. ↵ T. S. Lendvay , D. K. Morris , J. Sah , B. Balasubramanian , V. Lundblad , Senescence mutants of Saccharomyces cerevisiae with a defect in telomere replication identify three additional EST genes . Genetics 144 , 1399 – 1412 ( 1996 ). OpenUrl Abstract / FREE Full Text 59. ↵ R. J. Wellinger , V. A. Zakian , Everything you ever wanted to know about Saccharomyces cerevisiae telomeres: beginning to end . Genetics 191 , 1073 – 1105 ( 2012 ). OpenUrl Abstract / FREE Full Text 60. ↵ K. Myung , C. Chen , R. D. Kolodner , Multiple pathways cooperate in the suppression of genome instability in Saccharomyces cerevisiae . Nature 411 , 1073 – 1076 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 61. ↵ S. Kurtz , D. Shore , RAP1 protein activates and silences transcription of mating-type genes in yeast . Genes Dev 5 , 616 – 628 ( 1991 ). OpenUrl Abstract / FREE Full Text 62. ↵ B. Garvik , M. Carson , L. Hartwell , Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint . Mol Cell Biol 15 , 6128 – 6138 ( 1995 ). OpenUrl Abstract / FREE Full Text 63. ↵ C. I. Nugent , T. R. Hughes , N. F. Lue , V. Lundblad , Cdc13p: a single-strand telomeric DNA-binding protein with a dual role in yeast telomere maintenance . Science 274 , 249 – 252 ( 1996 ). OpenUrl Abstract / FREE Full Text 64. ↵ W. H. Chung , Z. Zhu , A. Papusha , A. Malkova , G. Ira , Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting . PLoS Genet 6 , 24 ( 2010 ). OpenUrl 65. ↵ J. Y. Hwang , S. Smith , K. Myung , The Rad1-Rad10 complex promotes the production of gross chromosomal rearrangements from spontaneous DNA damage in Saccharomyces cerevisiae . Genetics 169 , 1927 – 1937 ( 2005 ). OpenUrl Abstract / FREE Full Text 66. ↵ A. J. Bardwell , L. Bardwell , A. E. Tomkinson , E. C. Friedberg , Specific cleavage of model recombination and repair intermediates by the yeast Rad1-Rad10 DNA endonuclease . Science 265 , 2082 – 2085 ( 1994 ). OpenUrl Abstract / FREE Full Text 67. ↵ S. Boiteux , S. Jinks-Robertson , DNA repair mechanisms and the bypass of DNA damage in Saccharomyces cerevisiae . Genetics 193 , 1025 – 1064 ( 2013 ). OpenUrl Abstract / FREE Full Text 68. ↵ J. Sollier et al. , Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability . Mol Cell 56 , 777 – 785 ( 2014 ). OpenUrl CrossRef PubMed 69. ↵ T. Shi et al. , Rif1 and Rif2 shape telomere function and architecture through multivalent Rap1 interactions . Cell 153 , 1340 – 1353 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 70. ↵ E. A. Feldmann , R. Galletto , The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes . Biochemistry 53 , 7471 – 7483 ( 2014 ). OpenUrl CrossRef PubMed 71. ↵ Y. V. Le Bihan et al. , Effect of Rap1 binding on DNA distortion and potassium permanganate hypersensitivity . Acta Crystallogr D Biol Crystallogr 69 , 409 – 419 ( 2013 ). OpenUrl CrossRef 72. ↵ M. Costanzo et al. , A global genetic interaction network maps a wiring diagram of cellular function . Science 353 , aaf1420 ( 2016 ). 73. ↵ S. Ghaemmaghami et al. , Global analysis of protein expression in yeast . Nature 425 , 737 – 741 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 74. ↵ N. Y. Yao , A. Johnson , G. D. Bowman , J. Kuriyan , M. O’Donnell , Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C . J Biol Chem 281 , 17528 – 17539 ( 2006 ). OpenUrl Abstract / FREE Full Text 75. ↵ V. N. Noskov , H. Araki , A. Sugino , The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae . Mol Cell Biol 18 , 4914 – 4923 ( 1998 ). OpenUrl Abstract / FREE Full Text 76. ↵ K. Sugimoto et al. , Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast . Proc Natl Acad Sci U S A 93 , 7048 – 7052 ( 1996 ). OpenUrl Abstract / FREE Full Text 77. ↵ H. Tang , B. Hilton , P. R. Musich , D. Z. Fang , Y. Zou , Replication factor C1, the large subunit of replication factor C, is proteolytically truncated in Hutchinson-Gilford progeria syndrome . Aging Cell 11 , 363 – 365 ( 2012 ). OpenUrl CrossRef PubMed 78. ↵ M. Eriksson et al. , Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome . Nature 423 , 293 – 298 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 79. ↵ B. Cisneros , I. Garcia-Aguirre , M. De Ita , I. Arrieta-Cruz , H. Rosas-Vargas , Hutchinson-Gilford Progeria Syndrome: Cellular Mechanisms and Therapeutic Perspectives . Arch Med Res 54 , 102837 ( 2023 ). OpenUrl 80. ↵ M. L. Decker , E. Chavez , I. Vulto , P. M. Lansdorp , Telomere length in Hutchinson-Gilford progeria syndrome . Mech Ageing Dev 130 , 377 – 383 ( 2009 ). OpenUrl CrossRef PubMed 81. ↵ B. A. Kudlow , M. N. Stanfel , C. R. Burtner , E. D. Johnston , B. K. Kennedy , Suppression of proliferative defects associated with processing-defective lamin A mutants by hTERT or inactivation of p53 . Mol Biol Cell 19 , 5238 – 5248 ( 2008 ). OpenUrl Abstract / FREE Full Text 82. E. K. Benson , S. W. Lee , S. A. Aaronson , Role of progerin-induced telomere dysfunction in HGPS premature cellular senescence . J Cell Sci 123 , 2605 – 2612 ( 2010 ). OpenUrl Abstract / FREE Full Text 83. ↵ Y. Li et al. , Transient introduction of human telomerase mRNA improves hallmarks of progeria cells . Aging Cell 18 , e12979 ( 2019 ). OpenUrl 84. ↵ T. Kubota , Y. Katou , R. Nakato , K. Shirahige , A. D. Donaldson , Replication-Coupled PCNA Unloading by the Elg1 Complex Occurs Genome-wide and Requires Okazaki Fragment Ligation . Cell Rep 12 , 774 – 787 ( 2015 ). OpenUrl CrossRef PubMed 85. C. Yu et al. , Strand-specific analysis shows protein binding at replication forks and PCNA unloading from lagging strands when forks stall . Mol Cell 56 , 551 – 563 ( 2014 ). OpenUrl CrossRef PubMed 86. C. Johnson , V. K. Gali , T. S. Takahashi , T. Kubota , PCNA Retention on DNA into G2/M Phase Causes Genome Instability in Cells Lacking Elg1 . Cell Rep 16 , 684 – 695 ( 2016 ). OpenUrl CrossRef PubMed 87. ↵ K. Shemesh et al. , A structure-function analysis of the yeast Elg1 protein reveals the importance of PCNA unloading in genome stability maintenance . Nucleic Acids Res 45 , 3189 – 3203 ( 2017 ). OpenUrl 88. ↵ B. J. Merrill , C. Holm , The RAD52 recombinational repair pathway is essential in pol30 (PCNA) mutants that accumulate small single-stranded DNA fragments during DNA synthesis . Genetics 148 , 611 – 624 ( 1998 ). OpenUrl Abstract / FREE Full Text 89. ↵ A. S. Ivessa et al. , The Saccharomyces cerevisiae helicase Rrm3p facilitates replication past nonhistone protein-DNA complexes . Mol Cell 12 , 1525 – 1536 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 90. ↵ V. P. Schulz , V. A. Zakian , The Saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formation . Cell 76 , 145 – 155 ( 1994 ). OpenUrl CrossRef PubMed Web of Science 91. ↵ J. B. Boulé , L. R. Vega , V. A. Zakian , The yeast Pif1p helicase removes telomerase from telomeric DNA . Nature 438 , 57 – 61 ( 2005 ). OpenUrl CrossRef PubMed Web of Science 92. ↵ M. E. Budd , C. C. Reis , S. Smith , K. Myung , J. L. Campbell , Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta . Mol Cell Biol 26 , 2490 – 2500 ( 2006 ). OpenUrl Abstract / FREE Full Text 93. ↵ K. Paeschke , J. A. Capra , V. A. Zakian , DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase . Cell 145 , 678 – 691 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 94. ↵ K. Paeschke et al. , Pif1 family helicases suppress genome instability at G-quadruplex motifs . Nature 497 , 458 – 462 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 95. ↵ K. Myung , A. Datta , C. Chen , R. D. Kolodner , SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination . Nat Genet 27 , 113 – 116 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 96. ↵ M. Chang et al. , RMI1 / NCE4 , a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex . EMBO J 24 , 2024 – 2033 ( 2005 ). OpenUrl Abstract / FREE Full Text 97. ↵ C. Zhang , T. M. Roberts , J. Yang , R. Desai , G. W. Brown , Suppression of genomic instability by SLX5 and SLX8 in Saccharomyces cerevisiae . DNA repair 5 , 336 – 346 ( 2006 ). OpenUrl CrossRef PubMed 98. ↵ J. Oh , L. S. Symington , Role of the Mre11 Complex in Preserving Genome Integrity . Genes (Basel) 9 ( 2018 ). 99. ↵ K. A. Bernstein , S. Gangloff , R. Rothstein , The RecQ DNA helicases in DNA repair . Annu Rev Genet 44 , 393 – 417 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 100. ↵ B. Pfander , J. Matos , Control of Mus81 nuclease during the cell cycle . FEBS letters 591 , 2048 – 2056 ( 2017 ). OpenUrl CrossRef 101. ↵ Y. C. Chang , M. K. Oram , A. K. Bielinsky , SUMO-Targeted Ubiquitin Ligases and Their Functions in Maintaining Genome Stability . Int J Mol Sci 22 ( 2021 ). 102. ↵ I. Marcomini et al. , Asymmetric Processing of DNA Ends at a Double-Strand Break Leads to Unconstrained Dynamics and Ectopic Translocation . Cell Rep 24 , 2614 – 2628 e2614 ( 2018 ). OpenUrl 103. ↵ F. Sherman , Getting started with yeast . Methods Enzymol 350 , 3 – 41 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 104. ↵ D. C. Amberg , D. Burke , J. N. Strathern , D. Burke , L. Cold Spring Harbor, Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual ( Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y ., 2005 ). 105. ↵ S. E. Luria , M. Delbruck , Mutations of Bacteria from Virus Sensitivity to Virus Resistance . Genetics 28 , 491 – 511 ( 1943 ). OpenUrl FREE Full Text 106. ↵ C. D. Putnam , R. D. Kolodner , Determination of gross chromosomal rearrangement rates . Cold Spring Harb Protoc 2010 , pdb prot5492 (2010). 107. ↵ P. M. van Mourik et al. , Recombination-Mediated Telomere Maintenance in Saccharomyces cerevisiae Is Not Dependent on the Shu Complex . PLOS ONE 11 , e0151314 ( 2016 ). OpenUrl CrossRef 108. ↵ F. Supek , M. Bosnjak , N. Skunca , T. Smuc , REVIGO summarizes and visualizes long lists of gene ontology terms . PLoS One 6 , e21800 ( 2011 ). OpenUrl CrossRef PubMed References 1. C. D. Putnam et al. , Bioinformatic identification of genes suppressing genome instability . Proc Natl Acad Sci U S A 109 , E3251 – 3259 ( 2012 ). OpenUrl Abstract / FREE Full Text 2. M. Usaj et al. , TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network . G3 (Bethesda) 7 , 1539 - 1549 ( 2017 ). OpenUrl Abstract / FREE Full Text 3. A. Baryshnikova , Systematic Functional Annotation and Visualization of Biological Networks . Cell Syst 2 , 412 – 421 ( 2016 ). OpenUrl View the discussion thread. Back to top Previous Next Posted April 13, 2024. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms Fernando R. Rosas Bringas , Ziqing Yin , Yue Yao , Jonathan Boudeman , Sandra Ollivaud , Michael Chang bioRxiv 2024.04.11.589032; doi: https://doi.org/10.1101/2024.04.11.589032 Share This Article: Copy Citation Tools Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms Fernando R. 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