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Two Pif1 Family DNA Helicases Cooperate in Centromere Replication and Segregation in Saccharomyces cerevisiae
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Publication date (Electronic):
15 November 2018
Journal:
Genetics
Publisher:
Genetics Society of America
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Abstract
Pif1 family helicases are found in virtually all eukaryotes. Saccharomyces cerevisiae (Sc) encodes two Pif1 family helicases, ScPif1 and Rrm3. ScPif1 is multifunctional, required not only for maintenance of mitochondrial DNA but also for multiple distinct nuclear functions. Rrm3 moves with the replication fork and promotes movement of the fork through ∼1400 hard-to-replicate sites, including centromeres. Here we show that ScPif1, like Rrm3, bound robustly to yeast centromeres but only if the centromere was active. While Rrm3 binding to centromeres occurred in early to mid S phase, about the same time as centromere replication, ScPif1 binding occurred later in the cell cycle when replication of most centromeres is complete. However, the timing of Rrm3 and ScPif1 centromere binding was altered by the absence of the other helicase, such that Rrm3 centromere binding occurred later in pif1-m2 cells and ScPif1 centromere binding occurred earlier in rrm3 Δ cells. As shown previously, the modest pausing of replication forks at centromeres seen in wild-type cells was increased in the absence of Rrm3. While a lack of ScPif1 did not result in increased fork pausing at centromeres, pausing was even higher in rrm3 Δ pif1 Δ cells than in rrm3 Δ cells. Likewise, centromere function as monitored by the loss rate of a centromere plasmid was increased in rrm3 Δ but not pif1 Δ cells, and was even higher in rrm3 Δ pif1 Δ cells than in rrm3 Δ cells. Thus, ScPif1 promotes centromere replication and segregation, but only in the absence of Rrm3. These data also hint at a potential post-S phase function for ScPif1 at centromeres. These studies add to the growing list of ScPif1 functions that promote chromosome stability.
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Most cited references55
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DNA replication through G-quadruplex motifs is promoted by the Saccharomyces cerevisiae Pif1 DNA helicase.
Katrin Paeschke, John A Capra, Virginia Zakian (2011)
G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by noncanonical G-G base pairs. Genome-wide chromatin immunoprecipitation was used to determine the in vivo binding sites of the multifunctional Saccharomyces cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in vitro. G4 motifs were a significant subset of the high-confidence Pif1-binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, whereas non-G4 Pif1-binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication-stressed Pif1-deficient cells, which was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication, and stimulate DNA breakage. These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage. Copyright © 2011 Elsevier Inc. All rights reserved.
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Pif1 family helicases suppress genome instability at G-quadruplex motifs
Katrin Paeschke, Matthew Bochman, P. Daniela Garcia … (2013)
The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans. We show that exceptionally potent G-quadruplex unwinding is conserved amongst Pif1 helicases. Moreover, Pif1 helicases from organisms separated by >3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast. The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to novel genetic and epigenetic changes. Further, when expressed in yeast, human Pif1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.
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The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo
Cyril Ribeyre, Judith Lopes, Jean-Baptiste Boulé … (2009)
Introduction At the chromosomal level, in addition to coding regions and epigenetic modifications, the biological information also resides in DNA secondary structures, but this layer remains to be further deciphered. Biophysical and structural studies have long established that in vitro DNA can adopt diverse structures different from the canonical Watson-Crick conformations [1]. However, for a long time, the hypothesis that these structures occur in the native chromosomal context, as an integral part of the functional architecture of a chromosome, has been regarded with a certain skepticism. One example of such a non canonical DNA structure is the G-quadruplex, also named G-tetraplex or G4 DNA. These structures form in vitro in guanine-rich sequences that contain four tracts of at least three guanines separated by other bases, and are stabilized by G-quartets that form between four DNA strands [2]. Under physiological conditions, long runs of G4-forming sequences promote the formation of highly stable structures that can form spontaneously in vitro and, once formed, are very resistant to thermal denaturation. It is also important to consider that sequences that form G4-DNA slowly in vitro may be more prone to fold in vivo owing to the action of proteins that promote and/or stabilize their formation, such as the beta subunit of the ciliate Oxytricha telomere binding protein complex [3],[4]. Evidence for in vivo formation of G4 DNA has emerged in recent years. Notably, G4 DNA has been observed by electron microscopy from transcribed human G-rich DNA arrays in bacteria [5] and has been detected at the end of the ciliate Oxytricha telomeres by immunochemistry [6],[7]. As a complementary approach, genome-wide bioinformatic analyses have identified regions that have the potential to form G4 DNA within evolutionary diverse model systems, from bacteria to human. For example, in the human genome, more than 300,000 distinct sites have the potential to form G4 DNA [8],[9]. These sequences are highly over-represented in the promoter regions of diverse organisms, including human [10], yeast [11] and bacteria [12]. In addition, potential G4-forming sequences are found in G-rich arrays such as telomeres, rDNA or G-rich micro- and minisatellites. Hence, it has been suggested that their presence might affect transcriptional or post-transcriptional events when the G4 forming sequence is within the transcribed region [11],[13]. G4 DNA has also been proposed to participate in telomere capping, DNA replication and recombination [14]. However, it remains to be determined how and to what extent these secondary structures affect these processes and how they are maintained through DNA replication despite causing a structural impairment to the various nucleic acid processing enzymes. It is clear that DNA goes through a single strand configuration locally during processes like DNA replication, transcription or repair, and many models argue that this single stranded stage favors G4 DNA formation [14]. In vitro, several DNA helicases, such as the human BLM, WRN, FANCJ and the S. cerevisiae Sgs1, can unwind G4 structures. They preferentially unwind G4 DNA over partially duplex DNA, forked DNA or Holliday junction substrates, and their helicase activity is inhibited in presence of G4 DNA ligands [15]–[18]. In Caenorhabditis elegans the FANCJ homolog dog-1 is involved in the maintenance of G-rich regions by preventing intrinsic instability and loss of these regions [19],[20]. However, considering that different G-rich sequences can adopt very diverse secondary structures, and that in numerous instances genes encoding helicases are not essential, the questions of how many and which class of helicases are indeed able to process efficiently these secondary structures formed in guanine-rich regions in a given organism remains to be addressed. Also, until now, very few in vivo systems exist to study the involvement of helicases in processing these structures and assay artificially designed variant substrates. In the present study, which was aimed at characterizing the mechanism(s) of rearrangement of tandem DNA repeats, we uncover an unexpected function of the Pif1 helicase with regards to processing G4 structures. Pif1 is a member of a conserved family of 5′-3′ DNA helicases, with distant homology to the RecD bacterial helicase. The S. cerevisiae Pif1 protein is important both for maintenance of mitochondrial DNA [21],[22] and as a negative regulator of telomerase-mediated telomere lengthening [23],[24]. Here we report that Pif1 also affects stability of the G-rich CEB1 minisatellite when it is inserted into a yeast chromosome. In contrast, mutations in other helicases, including the S. cerevisiae RecQ homologue Sgs1, had no effect on CEB1 stability. In vitro, CEB1 formed G4 structures that were efficiently unwound by Pif1. Finally, mutation of the CEB1 repeats such that they were no longer able to form G4 structures made them insensitive to Pif1. Thus we demonstrated that one of the functions of the Pif1 helicase is to process G4 structures. As sequences with the ability to form G4 DNA are found throughout the yeast genome, beyond acting on intrinsically instable repeats, we propose that the processing of G4 structures by Pif1 may facilitate DNA replication, transcription and/or repair. Results The DNA Helicase Pif1 Actively Destabilizes CEB1 during Vegetative Growth We previously developed yeast strains to study the genetic instability of a natural 1.8 kb allele of the human minisatellite CEB1 inserted in the S. cerevisiae genome (Figure 1A). This allele (called CEB1-1.8) is composed of a tandem array of 42 polymorphic repeats of sizes varying between 36 and 43 base pairs (bp) [25] (Figure S1). In our standard assay, which measures the frequency of allele size variation after growth for seven generations at 30°C, approximately 0.3% of wild-type (WT) cells exhibit a change in CEB1 size (contractions and expansions). Using this system, we reported that CEB1-1.8 was strongly destabilized in the absence of the Rad27/FEN1 endonuclease (42% instability) [26]. 10.1371/journal.pgen.1000475.g001 Figure 1 The CEB1 minisatellite is unstable in pif1Δ deficient cells. (A) Structure of the genomic locus containing CEB1-1.8. (B) Southern blot analysis of CEB1-1.8 instability in haploid strains: WT (ORT2914), pif1Δ (ORT4843), pif1-K264R (ORT5083-4E). Each lane contains DNA extracted from pools of 12 independent colonies digested by AluI and hybridized with a CEB1-0.6 probe. (C) Structure of CEB1-1.8 rearrangements obtained in pif1Δ haploids. Each of the 42 CEB1-1.8 repeats is represented by a colored box and numbered (top). Nine rearrangements were sequenced and classified in three categories (1 to 3). The name of each rearranged allele is at the left. Hybrid repeats are represented by the two colors corresponding to the fused repeats. The white box in P23 indicates a motif that cannot be attributed to a specific parental motif. (D) CEB1 instability is Rad52 and Rad51 dependent. Southern blot analysis of CEB1-1.8 instability in pif1Δ rad52Δ (ORD7565-2C) and pif1Δ rad51Δ (ORD7574-9B) haploid strains. Same legends as in (B). Additional bands marked with an asterisk are presumably due to partial digestion by AluI. Recently, it was reported that the lethality caused by inactivation of the essential helicase/endonuclease Dna2, which participates with Rad27 in the maturation of Okazaki fragments, could be rescued by inactivation of the DNA helicase Pif1 [27]. These results prompted us to test if Pif1 also had an effect on the maintenance of CEB1 arrays in our system. Remarkably, in the absence of Pif1 (pif1Δ), the frequency of rearrangement by contractions or expansions of the parental allele increased 20-fold compared to WT cells (6% instability; Table 1, Figure 1B). As a control, a pif1Δ CEB1-1.8 strain containing a multicopy plasmid that expressed the WT PIF1 gene under the control of the PIF1 promoter did not exhibit CEB1 instability. Together, these results demonstrate that the absence of Pif1 destabilizes the CEB1-1.8 minisatellite at a rate of ∼1% per cell per generation. CEB1 instability was not specific to tracts inserted at the ARG4 locus as CEB1-1.8 inserted at the ADP1 locus in chromosome III was stable in the presence of Pif1 but was rearranged in its absence (3.6% instability; 7/192). The difference in stability between the two chromosomal locations is not statistically significant (Fisher's Exact test, p = 0.28). 10.1371/journal.pgen.1000475.t001 Table 1 Instability of CEB1-1.8 in haploid strains. Strain Genotype Number of rearrangements/total (%) Fold increase vs. WT p value vs. WT* ORT2914 WT 5/1824 (0.3) 1 - ORT4841 pif1Δ 40/672 (6.0) 20 1 kb) synthetic CEB1 alleles (see Text S1). We generated two categories of synthetic CEB1 arrays based on two different repeat units. The first category, named synthetic-CEB1-WT, was based on the repetition of the most common motif of the natural polymorphic CEB1-1.8 allele (Figure S3, A, D). The second category, named CEB1-Gmut, was made from oligonucleotides in which 5 dispersed G bases were changed to either C, A or T in order to disrupt the original 5 G-triplets on the G-rich strand (Figure S3, A, E). In vitro analysis of the secondary structures of CEB1-Gmut oligonucleotides demonstrated that, as expected, they were unable to form G4 structures (39Cebm, Figure 2 and Table S2). The rearrangement frequency of the synthetic-CEB1-WT arrays (1.0, 1.3, 1.7, 1.9 and 2.3 kb long) and of the synthetic-CEB1-Gmut arrays (0.7, 1.7, 2.5 and 3.8 kb long) in WT, pif1Δ and rad27Δ cells is reported in Table 3 and summarized in Figure 3. As observed for the natural CEB1 alleles, the rearrangement frequency of the synthetic-CEB1-WT arrays was low in WT cells and increased in a size dependent manner in both pif1Δ and rad27Δ cells. In all cases, the frequency of instability for similarly sized alleles was higher in the synthetic-CEB1-WT arrays than in the natural CEB1 alleles. We attribute this difference to the greatly reduced polymorphism of the synthetic allele. However, the most striking result was that mutations in G4 prone motifs strongly decreased the frequency of their rearrangement in pif1Δ cells. We observed only one rearrangement of the CEB1-Gmut-1.7 allele among the 383 colonies analyzed (0.2%) while the synthetic-CEB1-WT-1.7 allele was rearranged in 38/343 pif1Δ colonies (11%) (Figure 3 and Table 3). Similarly, the large synthetic-CEB1-Gmut-3.8 array, which contains approximately 97 repeats, yielded only a few rearrangements in the pif1Δ and WT strains (4% and 2%, respectively; this difference was not statistically different, p = 0.18, Fisher's Exact Test). In contrast, CEB1-Gmut arrays rearranged in rad27Δ cells and the frequency of rearrangement increased in a size dependent-manner (Table 3). Thus, the synthetic and natural CEB1 alleles behaved similarly while the artificial CEB1 arrays containing mutation of G4-prone sequences were stabilized in pif1Δ but not in rad27Δ cells. These results strongly support our proposal that formation of G4 structures within the CEB1 array is responsible for their instability in vivo and that this secondary structure is processed by the Pif1 helicase. 10.1371/journal.pgen.1000475.t003 Table 3 Instability of synthetic minisatellites in WT, pif1Δ and rad27Δ cells. Minisatellite Number of repeats Strain Genotype Number of rearrangements/total (%) CEB1-WT-2.3 58 AND1207-9B WT 6/384 (1.5) CEB1-WT-1.7 44 AND1212-10D WT 2/708 (0.3) CEB1-WT-1.0 26 AND1213-1D WT 0/192 (0) CEB1-WT-1.9 48 AND1202-13D-P14C3 pif1Δ 32/154 (20.6) CEB1-WT-1.7 44 AND1202-11A pif1Δ 38/343 (11) CEB1-WT-1.3 33 AND1202-11A-L8C12 pif1Δ 8/189 (4.2) CEB1-WT-1.0 26 ORT6108-4 pif1Δ 0/192 (0) CEB1-WT-1.7 44 AND1218-1A rad27Δ 15/50 (30) CEB1-WT-1.0 26 ORT6110-1 rad27Δ 32/576 (5.5) CEB1-Gmut-3.8 97 AND1206-5D WT 8/384 (2) CEB1-Gmut-1.7 42 AND1227-5C WT 0/192 (0) CEB1-Gmut-3.8 97 AND1206-4C pif1Δ 8/192 (4) CEB1-Gmut-2.5 64 AND1206-4C-1B6-1E1 pif1Δ 1/192 (0.5) CEB1-Gmut-1.7 42 AND1206-4C-D11P2 pif1Δ 1/383 (0.2) CEB1-Gmut-0.7 19 ORT6107-1 pif1Δ 0/192 (0) CEB1-Gmut-3.8 97 AND1206-4B rad27Δ 35/51 (68.6) CEB1-Gmut-1.7 42 AND1226-18B/-17C rad27Δ 52/98 (53) CEB1-Gmut-0.7 19 ORT6109 rad27Δ 23/552 (4.1) Discussion In the present study, we provide new insights into the biochemical and biological functions of the evolutionary conserved Pif1 helicase. Our main findings are: (i) inactivation of Pif1 increased the frequency of rearrangement of the G-rich CEB1-1.8 tandem array, (ii) this increased rearrangement was specific for Pif1 as mutation of other helicases did not affect the stability of CEB1 and other repeats were stable in pif1Δ cells, (iii) the G-rich strand of the CEB1 repeat unit formed G-quadruplex structures in vitro, (iv) Pif1 readily unwound the CEB1 G4 structures in vitro and, (vi) mutation of the G4–forming motifs stabilized CEB1 in pif1Δ cells. Destabilization of CEB1 in pif1Δ cells was not an indirect consequence of other pif1Δ phenotypes such as respiratory deficiency or long telomeres. Thus, the experiments reported here uncover a new activity for the Pif1 helicase, the ability to process G4 secondary structures, and suggest that this activity contributes to genome stability by preventing the rearrangement of G4 forming repeats in vivo. Mechanism of CEB1 Repeats Instability In previous studies, we reported that human CEB1 repeats inserted into the yeast genome are highly unstable in absence of the Rad27 endonuclease and slightly unstable in a dna2-1ts mutant [25],[26]. Since Rad27 and Dna2 are involved in the processing of flap structures during Okazaki fragment maturation [52], we concluded that CEB1 instability was likely due to the accumulation of unresolved flap structures during replication. We proposed that these intermediates would form recombinogenic structures that are repaired by homology-dependent strand displacement and annealing (SDSA) [53]. Here we show that inactivation of Pif1 also resulted in CEB1 instability. As in rad27Δ cells, the CEB1 rearrangements in pif1Δ cells had a high frequency of complex events (Figure 1C; [25]). In addition, in both mutants, CEB1 rearrangements depended on Rad52/Rad51-dependent homologous recombination (Table 1). These similarities suggest that the repair of the lesion leading to CEB1 rearrangement in the absence of either Pif1 or Rad27 occurs by SDSA, although the recombinogenic lesion may be different (for example a single-strand gap or a double-strand break). In pif1Δ and rad27Δ cells, the frequency of rearrangements increased with the size of the allele (Figure 3C; [25]). In rad27Δ cells, this increased instability may reflect the increased probability that longer arrays are more likely to contain more than one improperly processed flap. Similarly, in pif1Δ cells, long CEB1 minisatellites could form G4 structures with a higher probability, especially if quadruplexes involve G-tracts from adjacent repeats. Alternatively, lesions in small alleles could be rare or more often resected into the non-repeated flanking sequences, leading to the preferential restoration of the parental sequence by homologous recombination in G2 cells using the intact sister chromatid as a template [53]. CEB1 Repeats Are Unstable in pif1Δ Cells Only if They Are Able to Form G4 Structures Whereas all micro- and minisatellites sequences tested are unstable in rad27Δ cells ([43],[44] this study), only CEB1 was unstable in pif1Δ cells (Table 2). The CEB1 sequence is G/C rich (72%) with a high strand bias (23 G and 7 C per repeat of 39 bases). However, the instability of CEB1 in pif1Δ cells can not be attributed solely to its G/C rich sequence as the human hRAS1 minisatellite, which is also G rich (68%) with a strong bias (14 G and 5 C per repeat of 28 bases), was stable in the absence of Pif1. Each CEB1 repeat contains putative G4 signature motifs. Our biophysical analyses of CEB1 and hRAS oligonucleotides showed that the CEB1 motif readily formed G4 structures in vitro while hRAS1 did not (Figure 2 and Table S2). Moreover, synthetic CEB1 minisatellites in which the runs of guanine were mutated to disrupt their ability to form G4 structures were no longer unstable in pif1Δ cells. We propose that the recombinogenic lesions formed in the absence of Pif1 are unresolved intra- or inter-motifs G4 structures. Thus, while CEB1 alleles are unstable in both pif1Δ and rad27Δ cells, the events that initiate instability, unprocessed Okazaki fragments (in rad27Δ cells) or persistent G4 structures (in pif1Δ cells) are different (Figure 4). As a result, all tandem arrays are unstable in the absence of Rad27, including the synthetic G4-mutated CEB1 alleles, while only CEB1 was unstable in pif1Δ cells. 10.1371/journal.pgen.1000475.g004 Figure 4 Proposed model for CEB1 rearrangements in rad27Δ and pif1Δ mutants. In the absence of Rad27, accumulation of unresolved flap structures inside CEB1 during replication generates recombinogenic structures that are repaired by homology-dependent strand displacement and annealing (SDSA). While in the absence of Pif1, the persistence of unprocessed G-quadruplex secondary structures in CEB1, during replication, transcription or other processes, initiates DNA lesions that are also repaired by SDSA, leading to minisatellite rearrangements. In Vivo Roles of Pif1 What do our results suggest about the role(s) of Pif1 in the cell? Owing to the alternative use of a translation start site, PIF1 generates two isoforms, one with mitochondrial and one with nuclear functions. Several observations indicate that Pif1 is involved in the maintenance of mitochondrial DNA. Specifically, Pif1 increases the frequency of recombination between ρ+ and certain ρ− tandemly repeated mitochondrial genomes [21]. The loss of Pif1 is thought to trigger mtDNA breakage in specific regions, leading the authors to propose that Pif1 recognizes a specific but uncharacterized DNA topology [22],[54]. Although the ∼75 kb S. cerevisiae mitochondrial genome is AT-rich, it contains numerous G-rich stretches. We speculate that in the absence of mitochondrial Pif1, breaks occur due to defective processing of G4 structures and these breaks are repaired by recombination. Alternatively, G4 DNA can create a structural target for factors involved in DNA recombination. In the nucleus, Pif1 affects telomere length through direct inhibition of telomerase [23],[28] the specialized reverse transcriptase that lengthens telomeres in most eukaryotes. In vivo and in vitro data suggest that telomerase inhibition is achieved by direct displacement of telomerase from a DNA end [24]. Since Pif1 exhibits a marked preference for RNA-DNA hybrid unwinding in vitro [55], Pif1 is proposed to inhibit telomerase by unwinding the RNA-DNA hybrid formed between the telomerase RNA, TLC1, and the telomeric DNA end. Pif1-mediated removal of telomerase from DNA ends can explain the effects of pif1 mutations on both telomere length and de novo telomere addition [23],[56] as well as its inhibition of gross chromosomal rearrangements [57]. Human Pif1 (hPIF) may have similar functions as ectopic expression of hPIF causes telomere shortening and decreased telomerase processivity in vitro [58]. In addition, hPIF co-immunoprecipitates with telomerase subunits and telomerase activity [59]. Importantly for the present study, most telomeric DNA sequences, including yeast and human telomeric DNA, can form G4 structures in vitro. Moreover, G4 structures have been detected at ciliate telomeres in vivo [6]. In budding yeast, no evidence of the presence of G4 structures in the telomeric single stranded region has yet been reported, but proteins that bind or process G4 DNA in vitro are nevertheless present at yeast telomeres. In particular, in vitro studies have shown that the telomere binding protein Rap1 binds double-stranded telomeric DNA and promote the formation of G-quadruplex structures [60]. It is not known if this reaction occurs in vivo, but it is tempting to speculate that the formation of G4 DNA is necessary to promote the assembly of functional telomere. Alternatively or in addition to its ability to inhibit telomerase directly, Pif1 could counteract the formation of G4 structures in telomeric DNA, thus antagonizing the formation of proper telomere architecture. Consistent with this hypothesis, it has been shown that Pif1 overexpression compromises the viability of yeast strains with compromised telomere end protection [61]. Several studies suggest that Pif1 also has non-telomeric roles in replication and repair of nuclear DNA. First, in the rDNA, Pif1 helps maintain the replication fork barrier during replication [32]. Second, Pif1 is recruited to Rad52 DNA repair foci after gamma irradiation [62]. Third, lack of Pif1 suppresses the lethality of a Dna2 deletion, a helicase/endonuclease involved in the processing of Okazaki fragments by removing long 5′ flaps. Although the role of Pif1 in Okazaki fragment maturation is unclear, it is proposed to act by extending the flaps created by the lagging strand replicative polymerase at the junction of two consecutive Okazaki fragments [27]. Like Pif1, Dna2 is involved in telomere maintenance [63] and is able to process G4 DNA in vitro [64]. Thus, the two enzymes may act in concert to remove toxic intermediates, including G4-DNA, which could arise during lagging strand replication and, if not appropriately processed, promote formation of recombinogenic DNA lesions, such as double strand breaks. Finally, considering that in addition to G4-unwinding, Pif1 more efficiently unwinds RNA/DNA hybrids than DNA/DNA substrates [55], it is also to be envisaged that Pif1 plays a more general role in yeast cells when potential G4 structure can form, for example, during transcription. Multiplicity and Specificity of G4-Processing Helicases Budding yeast as well as all the other organisms encodes a large number of helicases. Current estimate in S. cerevisiae is approximately 120. This multiplicity raises the question of their specific substrate(s) and function(s), an issue which remains often unresolved and controversial. In S. cerevisiae, the RecQ homolog Sgs1 helicase was proposed to resolve G4 DNA, a conclusion primarily based on its ability, and more generally of members of the RecQ family, to resolve G4 DNA structures in vitro [16]. Compelling evidence for the involvement of Sgs1 in G4 DNA metabolism in vivo finally came from the survey of global gene expression analysis in absence of Sgs1 [11]. The authors found that the set of genes which expression level is affected in sgs1 mutant is biased towards genes that contain potential G4 forming sequences in their ORFs. To our surprise, the deletion of SGS1 had no effect on CEB1 stability (Table 1). The lack of in vivo redundancy between Sgs1 and Pif1 in this novel assay is interesting and allows several hypotheses. First, it is possible that Sgs1 and Pif1 do not recognize the same set of G4 structures. G4 forming sequences can give rise to secondary structures exhibiting very diverse sizes, topologies (parallel or anti-parallel) and arrangements (intra- or inter-molecular) [65], and these structures may be recognized or processed differently depending on helicase. Second, Sgs1 may not recognize the G4 substrates generated by CEB1 in vivo due to the polarity of the single strand region flanking the G4-DNA structure (Pif1 is a 5′-3′ helicase while Sgs1 has a 3′-5′ polarity). Third, it is likely that the numerous repeats in CEB1 that contain G4 forming sequences lead to the formation of highly stable structures in vivo that only some helicases are able to unwind. Finally, in the absence of more direct evidences for Sgs1 involvement in G4 DNA unwinding in vivo, there is also a possibility that Sgs1 plays a minor role in maintaining G4 DNA forming sequences. In multicellular organisms, the relationships between genomic instability, G-quadruplex structures and helicases functions have also been suspected. Studies in human cells deficient for the Werner, Bloom and RTEL helicases showed defects in telomere maintenance in vivo while G4 DNA is highly suspected to form at mammalian telomeres [66],[67] and a recent study reports the correlation between genomic stability and G4 DNA unwinding by the human FANCJ helicase [18]. Similarly, in Caenorhabditis elegans, the disruption of the RTEL homolog DOG-1 triggers deletions of polyguanine tracts matching the G4 DNA signature [20]. Finally, it should be mentioned that the inactivation of the potential Pif1 homolog in mice has no detectable phenotype, in particular regarding change in telomere length homeostasis [68]. In light of our present study, the stability of other repeated potentially G4 forming sequences in mice and mammalian cells should be examined. Also, taking advantage of the present yeast system allowing to test natural and synthetic substrates, we anticipate that further studies of pif1Δ cells will allow to uncover the multiple roles of this evolutionary conserved helicase, facilitate the characterization of G4 structures in vivo and finally enhance our understanding of the dynamics of G4 formation and function in vivo. Materials and Methods Yeast Strains The relevant genotypes and sources of haploid and diploid S. cerevisiae strains (S288C background) used in this study are indicated in Table S1. Identification of Minisatellite Rearrangements Examination of CEB1 instability during vegetative growth was done as previously described [25]. Individual colonies or colonies pools were analyzed by Southern blot depending on the rearrangement frequency (for rearrangement frequency >20%, individual colonies were privileged). Southern blots were performed using AluI digestion for natural CEB1 minisatellites and ApaI/SpeI for synthetic minisatellites and the corresponding membranes were hybridized with the radiolabeled CEB1-0.6 and CEB1-synthetic probes, respectively. For the analysis of the yeast minisatellite instability (DAN4, FLO1, HKR1 and NUM1), Southern blots were performed using AluI digestion (which does not cut in these repeats). Membranes were hybridized with the radiolabeled purified PCR product of the corresponding minisatellite (primer sequences available under request). For the analysis of the human hRAS1 minisatellite instability, Southern blots were performed using ApaI/SpeI digestion and hRAS1 probe obtained from the p37Y8 plasmid (gift from D. Kirkpatrick). Detection of signals was done with a Storm PhosphorImager (Molecular Dynamics). For pools of genomic DNA from 12 or 16 colonies/wells, rearrangement is counted when the intensity of the rearranged minisatellite, quantified with ImageQuant software, corresponds to 1/12 or 1/16 of the total amount of signals measured in the lane. When several rearranged minisatellites migrate at the same size they are considered as clonal and are counted only once. Sequencing of CEB1 Alleles The internal structure of rearranged alleles was determined by DNA sequencing as described previously [25]. Analysis of G-Quadruplex Secondary Structure Oligonucleotides were synthesized by Eurogentec (Belgium). Concentrations of all oligodeoxynucleotides were estimated using extinction coefficients provided by the manufacturer and calculated with a nearest neighbor model [69] under low salt conditions at 60°C in order to destabilize quadruplex formation. The sequences studied are shown in Table S2. Oligonucleotides chosen for non denaturing gel electrophoresis were first purified under denaturing conditions. Melting experiments were conducted as previously described [70]. Denaturation was followed by recording the absorbance at 240 or 295 nm [47],[71]. Melting experiments were typically performed at a concentration of 4 µM per strand. Thermal difference spectra (TDS) were obtained by difference between the absorbance spectra from unfolded and folded oligonucleotides that were respectively recorded much above and below its melting temperature (Tm). Circular dichroism (CD) spectra were recorded on a JASCO-810 spectropolarimeter using a 1 cm path length quartz cuvette in a reaction volume of 580 µl. Oligonucleotides were either i) prepared as a 4 µM solution in 10 mM lithium cacodylate pH 7.2, 100 mM NaCl or KCl buffer and annealed by heating to 90°C for 2 min, followed by cooling to 20°C or ii) preincubated for 48 hours at higher strand concentration (140 µM) in a 10 mM lithium cacodylate pH 7.2, 1 M NaCl buffer. Scans were performed at 25°C to 90°C over a wavelength range of 220–335 nm with a scanning speed of 500 nm/min, a response time of 1 s, 1 nm pitch and 1 nm bandwidth. Formation of G4-DNA was confirmed by non-denaturing PAGE. In this case, oligonucleotides were either directly observed by UV shadow (when incubated at high strand concentration) or 5′ labeled with T4 polynucleotide kinase. Prior to the incubation, the DNA samples were heated at 90°C for 10 min and slowly cooled (2 h) to room temperature (or 60°C for 48 hours). Oligonucleotides were first treated with 50 mM LiOH (to unfold quadruplexes) for 10 minutes followed by HCl neutralization. Samples were incubated at 10 nM or 4 µM strand concentration in Tris-HCl 10 mM pH 7.5 buffer with 100–1000 mM Li+ or K+. 10% sucrose was added just before loading. Oligothymidylate markers (dT15, dT21, or dT30) or double-stranded markers (Dx9: 5′d-GCGATACGG+5′d-CCGATACGC Dx12: 5′d-GCGTGACTTCGG+5′d-CCGAAGTCACGC) were also loaded on the gel. Analysis of G-Quadruplex Unwinding by Pif1 In Vitro Recombinant Pif1 was purified to homogeneity by affinity chromatography as described [55]. A Cy5-labeled oligonucleotide containing a 5′ poly(dA) tail followed by a CEB1 repeat (5′-Cy5-AAAAAAAAAAAGGGGGAGGGAGGGTGGCCTGCGGAGGTCCCTGGGCTG) was synthesized by Eurogentec (Belgium). For formation of the G-quadruplex, a solution of CEB1 oligo at 140 µM in 1 M NaCl was denatured 5 min at 100°C, then incubated at 65°C for 48 hours to promote formation of G4 intermolecular structures [72]. The double-stranded DNA control was made by annealing a 5′-Cy5-labeled 20 mer oligonucleotide to a 40 mer oligonucleotide, leaving a 20 nucleotide-long 5′ single-stranded DNA overhang. Briefly, 10 µM of each oligonucleotide were mixed in a buffer containing 10 mM Tris pH 8.0 and 5 mM Mg2+. The mixture was denatured 5 minutes at 95°C and slowly let to cool to room temperature. The double-stranded DNA substrate was further purified from non annealed single-stranded DNA on a MiniQ anion exchange column. Helicase assays were carried out by incubating indicated amounts of Pif1 and 2 nM nucleic acid substrate at 35°C. Standard reaction buffer was 20 mM Tris pH 7.5, 50 mM NaCl, 100 µg/ml bovine serum albumin, 2 mM DTT, 5 mM Mg2+ and 4 mM ATP. For kinetic studies, reactions were started by addition of ATP in presence of 100 nM Pif1 and 2 nM substrate. 10 µl aliquots were withdrawn at indicated times and the reactions stopped by addition of 2 µl deproteinizing/loading buffer (6% Ficoll, 50 mM EDTA pH 8.0, 2.5 µg/µl Proteinase K) and incubated further 15 minutes at 35°C. Reaction products were loaded on a 10% polyacrylamide non-denaturing gel and resolved by electrophoresis at 4°C and 10 V/cm in TBE 1× buffer. Gels were dried and scanned with a storm PhosphorImager (Molecular Dynamics) and quantified using ImageQuant software (GE Healthcare). Statistical Analysis Fisher exact test was performed using R software [73]. Supporting Information Figure S1 Sequences of the G-strand of CEB1-1.8 parental allele and of nine rearrangements obtained in the pif1Δ haploid strain (ORT4841). Polymorphic DNA bases are highlighted. The numbers at right in parentheses indicate the corresponding repeat in the parental CEB1-1.8 allele. Two numbers separated by dash represent hybrid repeats. Junction regions, which are delimited by polymorphisms of CEB1-1.8 derived from repeats involved in the deletions/duplications, are shaded in grey. X indicates a repeat of unknown origin or which cannot be attributed to a specific repeat in the parental CEB1-1.8 allele. (1.66 MB PDF) Click here for additional data file. Figure S2 Behavior of the 39Ceb and 39Cebm sequences on a non-denaturing gel. Two strand concentrations were tested: radiolabeled only (around 50 nM) or supplemented with 4 µM of cold oligonucleotide. Samples were treated with 50 mM LiOH to unfold quadruplexes, reannealed in 1 M NaCl buffer (top) or KCl (bottom) for 2 hours and loaded on a non-denaturing 15% acrylamide gel and run at 26°C. Migration markers are double-stranded DNA (9 and 12 bp) and (dT)15, (dT)21 and (dT)30 oligomers. (0.11 MB PDF) Click here for additional data file. Figure S3 Synthesis of artificial CEB1 minisatellites by PCR. (A) Nucleotide sequence of the CEB1-WT and CEB1-Gmut motifs. Repeats of at least three consecutive guanines are highlighted in grey in the CEB1-WT motif. Point mutations interrupting the G-triplets in the CEB1-Gmut motif are underlined. (B) Schematic representation of CEB1-concatemers synthesized by PCR. Two complementary oligonucleotides for CEB1-Gmut are represented (up and low), each composed of two identical CEB1-Gmut motifs (see Text S1 for sequences). After the first cycle of denaturation and annealing, the oligonucleotides can perfectly anneal along the two motifs and no elongation is possible (left), or they can shift and only one motif is annealed and the second motif is used as DNA template for elongation (right) resulting in addition of one motif at the end of the cycle. (C) After 30 cycles, DNA is deposited in agarose gel and the smear corresponds to a population of CEB1-concatemers of various sizes. White square indicates the part of the gel that will be cut in order to extract DNA and clone it in pGEM-T Easy vector. Sequences of the synthetic minisatellites, CEB1-WT-1.0 (D) and CEB1-Gmut-1.7 (E), with 26 and 42 repeats respectively. The sequence of the parental motif (CEB1-WT or CEB1-Gmut) used for the synthesis is indicated above the sequence of the synthetic minisatellite. Mutations and small deletions introduced during the concatemer synthesis are highlighted in red and in grey, respectively. (0.51 MB PDF) Click here for additional data file. Table S1 List of strains used in this study. (0.10 MB PDF) Click here for additional data file. Table S2 Sequence of the oligonucleotides used and their respective melting temperatures. (0.08 MB PDF) Click here for additional data file. Text S1 Supplementary material and methods. (0.13 MB PDF) Click here for additional data file.
Author and article information
Journal
Journal ID (nlm-ta): Genetics
Journal ID (iso-abbrev): Genetics
Journal ID (hwp): genetics
Journal ID (pmc): genetics
Journal ID (publisher-id): genetics
Title:
Genetics
Publisher:
Genetics Society of America
ISSN
(Print):
0016-6731
ISSN
(Electronic):
1943-2631
Publication date
(Print):
January
2019
Publication date
(Electronic):
15
November
2018
Publication date PMC-release: 15
November
2018
Volume: 211
Issue: 1
Pages: 105-119
Affiliations
Author notes
[1
]Corresponding author: Department of Molecular Biology, Washington Rd., Lewis Thomas
Laboratory, Princeton University, Princeton, NJ 08544. E-mail:
vzakian@
123456princeton.edu
Article
Publisher ID: 301710DOI: 10.1534/genetics.118.301710
PMC ID: 6325707
PubMed ID: 30442759
SO-VID: ae933d71-4c2a-4156-90b5-55e614e60c95
Copyright © Copyright © 2019 by the Genetics Society of America
License:
Available freely online through the author-supported open access option.
History
Date
received
: 17
August
2018
Date
accepted
: 06
November
2018
Page count
Figures: 6,
Tables: 1,
Equations: 0,
References: 61,
Pages: 15
Categories
Subject:
Investigations
Subject:
Genome Integrity and Transmission
ScienceOpen disciplines: Genetics