Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner.

The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the structure and activities of Pot1 in Ustilago maydis, a fungal model that recapitulates key features of mammalian telomere regulation. Compared to the well-characterized primate and fission yeast Pot1 orthologs, UmPot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. UmPot1 binds directly to Rad51 and regulates the latter's strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere maintenance. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1-deficient cells were suppressed, and cell viability was restored by the deletion of genes encoding Rad51 or Brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection.

Introduction

A key function of telomeres is to prevent chromosome ends from being recognized as double strand breaks (DSBs); erroneous recognition of telomeres as DSBs leads to the activation of DNA damage response (DDR), resulting in detrimental DNA repair (i.e., non-homologous end joining (NHEJ) or homology-directed repair (HDR)) that engenders chromosomal re-arrangements [1,2]. This protective function of telomeres is largely mediated by the shelterin complex, a six-protein assembly that coats double-stranded telomere DNA repeats as well as the terminal 3’ overhang (G-overhang) [1]. Among the components of shelterin are proteins that directly recognize double-stranded telomere DNA (TRF1 and TRF2), one that recognizes the G-overhang (POT1), and several associated and bridging factors (RAP1, TIN2, and TPP1). The mechanisms by which shelterin components suppress DDR signaling and DNA repair pathways and the division of labor between these components remain central questions in telomere research.

The second key function of telomeres is to promote the retention and replenishment of telomere DNA, which is subject to two types of erosion mechanisms: 1) abrupt truncation due to failure of replication forks to traverse the G/C-rich telomere repeats or DNA damage and recombination; and 2) gradual erosion due to the failure of lagging strand DNA synthesis to copy the parental DNA strand in its entirety (“end replication problem”). This DNA maintenance function of telomeres is also partly mediated by shelterin. However, in contrast to the protective function, which entails suppression of DNA repair, the DNA maintenance function often entails positive regulation of DNA repair factors or polymerases by shelterin components. For example, TPP1 serves a critical function in recruiting telomerase, a special reverse transcriptase that compensates for telomere loss by extending the G-strand [3-5]. Similarly, mammalian TRF1 is believed to recruit the BLM helicase to telomeres to help unwind G-rich secondary structures, thereby promoting replication fork progression [6]. Thus, shelterin components evidently regulate the DNA repair machinery in a context-dependent manner in order to mediate both telomere protection and telomere DNA maintenance. Understanding the molecular basis of such dichotomous regulation is a major challenge in telomere research.

The G-strand binding protein Pot1 is one component of shelterin that plays multi-faceted roles in telomere protection and telomere DNA maintenance. Regarding telomere protection, current evidence indicates that mammalian POT1 selectively represses ATR signaling as well as abnormal telomere repair, including HDR and alternative NHEJ (a-NHEJ) [7-11]. However, in fission yeast, the loss of Pot1 leads to rapid telomere loss, culminating in the circularization of chromosomes through NHEJ [12,13]. Why the loss of Pot1 results in disparate outcomes is not well understood. POT1 orthologs have also been implicated in telomere DNA maintenance. Mouse POT1b (one of the two POT1 homologs in this organism) interacts with the cognate CTC1-STN1-TEN1 complex (CST) to promote telomere lagging strand synthesis and maturation [14]. CST is another widely conserved telomere complex that promotes telomere maintenance in parallel with shelterin by regulating primase-Pol α [15-17]. Human POT1 mutations that disrupt the POT1-CST interaction also trigger telomere replication defects [18].

An attractive model for dissecting the interplay between telomere proteins and DNA repair factors is the yeast-like basidiomycete fungus Ustilago maydis, originally developed by Robin Holliday to investigate recombinational repair [19]. In comparison to budding and fission yeasts, U. maydis bears greater resemblances to mammalian cells with respect to the telomere and HDR repair machinery, suggesting that it may be a more accurate model for mammalian telomere regulation [20,21]. U. maydis also harbors the canonical 6-bp telomere repeat tracts ([TTAGGG]n/[CCCTAA]n), which are ~500 bp long at each chromosome end [22]. Indeed, we showed that several repair proteins in U. maydis promote telomere replication and telomere recombination just like their mammalian orthologs [23-25]. Accordingly, we have taken advantage of U. maydis to examine the mechanisms and regulation of repair proteins at telomeres [23-26]. In one of the published studies, we show that the two double strand telomere binding proteins in U. maydis, named Tay1 and Trf2, play distinct roles in telomere maintenance and protection. Tay1, in particular, physically interacts with the Blm helicase and acts in conjunction with the latter to promote telomere replication, providing one illustration of the significance of telomere-repair protein interactions [27].

Herein we report our investigation of U. maydis Pot1, the conserved and essential G-strand binding protein. Compared to the well characterized mammalian and fission yeast Pot1 orthologs, UmPot1 harbors an extra N-terminal OB fold domain (OB-N), which was recently shown to be conserved in most metazoan Pot1 proteins [28]. Functional analysis in U. maydis revealed multi-faceted, Pot1-dependent effects of DNA repair factors on telomere regulation. Specifically, we showed that Pot1 binds directly to Rad51 and regulates the strand exchange activity of Rad51. Deleting the OB-N domain, which is partly responsible for Rad51-binding, caused telomere shortening, suggesting that Pot1 positively regulates the telomere replication function of Rad51. However, when Pot1 is depleted through transcriptional repression and telomeres become deprotected, Rad51 and Brh2 act to instigate aberrant repair and the production of abnormal telomere structures, including high levels of telomere repeat-containing non-coding RNAs (i.e., TERRA and its complement). Thus, Pot1 also negatively regulates the activities of Rad51 and Brh2 to suppress telomere instability. Together, these findings illuminate the evolution of Pot1 and demonstrate how a single telomere protein can regulate the HDR machinery in disparate manners in the contexts of normal and deprotected telomeres.

Results

UmPot1s in Basidiomycetes harbor an extra N-terminal OB fold that exhibits no nucleic acid-binding activity

Like the well characterized fission yeast and primate orthologs, UmPot1 possesses three conserved OB fold domains (OB1, OB2 and OB3) and exhibits high affinity binding to the canonical G-strand repeats [26]. However, UmPot1 also contains a long N-terminal extension (~ 350 amino acids before OB1) of unknown function (Fig 1A). To gauge the extent of conservation for this region, we used BLASTp or PSI-BLAST to identify additional fungal Pot1 homologs both within and beyond the Basidiomycota phylum. Notably, the majority of basidiomycete Pot1s contain an equivalent N-terminal extension (~200–350 amino acids (a.a.)) before OB1 (Fig 1A). Sequence alignment coupled with secondary structure prediction revealed a moderately conserved 120 a.a. domain exhibiting structural features of an OB-fold, i.e., five β strands with an intervening α helix (Fig 1B) [29]. Thus, most basidiomycete Pot1s appear to harbor four OB-fold domains (designated OB-N, OB1, OB2 and OB3). Interestingly, Myler et al. recently showed that this is true for the majority of metazoan POT1s as well [28]. In support of common ancestry, we found that the two groups of OB-Ns from basidiomycetes and metazoan Pot1s align well to each other (S1A Fig). In addition, homology modeling of a basidiomycete Pot1 OB-N (from PaPot1) yielded a structure that is superimposable on the OB-A domain from human RPA70 (Fig 1C). Similarities between metazoan and basidiomycete Pot1s are further underscored by the conservation of zinc-binding motifs in the OB3 of these homologs (S1B and S1C Fig). Interestingly, in contrast to basidiomycetes, the Pot1 homologs in other fungal phyla (e.g., Ascomycota, Chytridiomycota, Zoopagomycota, etc.) contain just three OB folds (Fig 1A). Because phylogenomic analysis indicates that Chytridiomycota and Zoopagomycota represent deeper fungal lineages than Basidiomycota, it is possible that OB-N might have been independently lost in multiple fungal branches [30], just as it was lost in selected metazoans [28]. Alternatively, basidiomycete Pot1 might have acquired OB-N through the duplication of another OB fold in the protein, such OB1. However, phylogenetic comparison suggests that the OB-N of basidiomycetes is more closely related to the OB-N of metazoans than to the OB1 of basidiomycetes (S2A Fig). Likewise, the same analysis failed to uncover a strong kinship between the OB-N and OB2 of basidiomycete Pot1 (S2B Fig). In addition, given the identical domain organization for the 4-OB Pot1 and RPA1, we assessed the relationship between the OB-N of Pot1 and RPA70 (S2C Fig). Again, the OB-N of metazoan and basidiomycete Pot1 are more similar to each other than to the OB-N of RPA70. Therefore, we favor the notion of a common origin between metazoan and basidiomycete Pot1 OB-N. However, we cannot rule out the possibility that the OB-N of basidiomycete Pot1 was acquired independently and came to resemble its metazoan counterpart through convergent evolution.

Fig 1

U maydis Pot1 and other basidiomycetes harbor an extra N-terminal OB fold domain.

A. Domain organization of Pot1 homologs from major fungal branches. B. Multiple sequence alignment of the N-terminal region of Basidiomycete Pot1 homologs. The alignment and secondary structure predictions were generated using PROMALS3D and displayed using MView. C. A homology model of the putative OB-N domain from Paxillus ammoniavirescens (PaPot1) (generated from SWISS-MODEL) is superimposed on the OB-A domain of human RPA70. Note that the five beta strands (labeled as S1 to S5) in the two structures are spatially well aligned. D. EMSA analysis of the DNA-binding activities of UmPot1 truncation variants. The domains included in each variant are illustrated at the top and the binding assays shown at the bottom. 5’-labeled G4 oligonucleotide was used as the probe.

In contrast to OB-N, the OB3 domain is evidently well conserved in all fungal phyla, except for the loss of Zinc-binding residues in ascomycetes Pot1 (S1C Fig). We conclude that relative to other fungal Pot1s, basidiomycete Pot1 may be structurally closer to the majority of metazoan orthologs.

To assess the contribution of OB-N as well as other domains of UmPot1 to DNA binding, we purified truncation variants of Pot1 and analyzed their G-strand binding activity (Figs 1D and S3A and S3B). Consistent with results for fission yeast and mammalian Pot1 orthologs, we found that high affinity G-strand-binding was mediated exclusively by the OB1 and OB2 of U. maydis Pot1. In contrast, neither the OB-N nor OB3 exhibited any appreciable binding activity (Fig 1D). These results suggest that OB-N may mediate its function through protein-protein interactions.

UmPot1 physically interacts with Rad51 and stimulates the strand exchange activity of Rad51

Since our overall objective in studying U. maydis telomeres was to dissect the interplay between telomeres and repair proteins, we screened for potential interactions between Pot1 and DNA repair proteins. Using FLAG-tagged Pot1 in affinity pull down assays, we detected substantial binding of Pot1 to purified Rad51, but not Brh2 (Fig 2A). This binding is probably direct (i.e., not through contaminating nucleic acids) because the addition of benzonase had no effect (Fig 2B). We then examined the Rad51-binding activities of different domains of Pot1 and detected significant activity for OB-N and OB12 of Pot1, but minimal binding for OB3 (S3C Fig). A Pot1 fragment that spans the N-terminus and OB12 showed stronger binding to Rad51 than either domain alone, further supporting the contributions of both regions to Rad51 interaction.

Fig 2

U maydis Pot1 binds to Rad51 and regulate the strand-exchange activity of Rad51.

A. Anti-FLAG agarose resin was pre-bound with Pot1-FLAG and tested for interaction with purified Rad51 or MBP-Brh2/Dss1 complex. The bound proteins were eluted with FLAG3 peptides and the levels of Rad51, Brh2 and Pot1 in the eluates analyzed using anti-Rad51, anti-MBP, and anti-FLAG antibodies. B. The interaction between Pot1-FLAG and Rad51 was analyzed in the absence or presence of Benzonase. C. The ability of Rad51 to mediate strand exchange between a 70-mer ssDNA and a labeled 39-mer dsDNA was analyzed in the absence or presence of ATP. The substrates and products are marked by an open and a closed arrowhead, respectively. Asterisks designate the positions of the 5’ 32P label. D. The effects of Pot1 and Tay1 on strand exchange were analyzed in the absence or presence of Rad51. The substrates and products are marked by an open and a closed arrowhead, respectively. Asterisks designate the positions of the 5’ 32P label. An additional band is detected in Pot1-containing reactions (marked by a filled circle). While the identity of this is unclear, it is not Rad51-dependent and unlikely to be relevant to the strand-exchange reaction.

To determine if Pot1 can modulate the recombinase activity of Rad51, we employed an oligonucleotide-based strand exchange assay [31,32]. As expected, Rad51 alone catalyzed strand exchange between the duplex and ssDNA substrates in an ATP-dependent manner (Fig 2C). Interestingly, while Pot1 alone exhibited no strand exchange activity, it stimulated strand exchange by ~ 2-fold in the presence of Rad51 (Fig 2D). By contrast, the duplex telomere protein Tay1, which does not bind Rad51 [27], had no effect on strand exchange either in the absence or presence of Rad51 (Fig 2D). We further examined the ability of Pot1 truncation derivatives to stimulate strand exchange, and found that both the N-terminus and OB12 domains were required for stimulation (S3D Fig). Together, these results implicate direct Pot1-Rad51 interaction in the stimulatory effect of Pot1. It is worth noting that the substrates in the strand exchange assays do not harbor telomere repeats (S2 Table) and are not expected to bind Pot1, again suggesting that the effect of Pot1 is mediated through protein-protein interaction rather than DNA-binding.

UmPot1 promotes telomere maintenance

The Pot1-Rad51 interaction suggests that the telomere maintenance function of Rad51 [24,26] might be regulated by Pot1. To test this idea, we sought to determine the consequence of disrupting this interaction. Because mutating the OB12 domain will likely abrogate DNA-binding and trigger telomere deprotection (making it difficult to interpret the phenotypes), we deleted just the OB-N of Pot1 (pot1ΔN) and used this allele to replace genomic pot1 through homologous recombination. The pot1ΔN mutant did not exhibit obvious growth defect or UV sensitivity (Fig 3A), suggesting that telomere protection is intact and Pot1 does not affect the genome-wide repair function of Rad51. In addition, pot1ΔN did not exhibit growth defects or evidence of senescence even following extensive passage on plates (Fig 3B). We then examined telomere maintenance of pot1ΔN using Southern analysis of telomere restriction fragments (TRF), and detected a moderate telomere length reduction in later passages of the mutant in comparison to the wild type strain (Fig 3C, streaks 6 and 10). To obtain a more quantitative assessment of telomere lengths, we used STELA (single telomere length analysis) to characterize the subpopulations of telomeres that harbor the UT4 subtelomere sequence [22]. Consistent with Southern analysis of TRF lengths, the STELA products in pot1ΔN were ~ 200 bp shorter than wild type at streak 10, supporting the notion that Pot1 promotes telomere maintenance (Fig 3D). To further test the notion that Pot1-Rad51 interaction underpins the replication function of Pot1, we compared the telomeres of the rad51Δ pot1ΔN double mutant to those of rad51Δ and pot1ΔN single mutants (S4A Fig). No significant differences were observed, supporting the idea that Rad51 acts in the same pathway as the OB-N of Pot1 to promote telomere maintenance. We also explored the effect of replication stress on the telomere phenotype of pot1ΔN. However, even low concentrations of hydroxyurea induced substantial telomere length heterogeneity in the wild type strain and there was no obvious exacerbation of telomere defects in pot1ΔN (S4B Fig).

Fig 3

Pot1 N-terminal deletion impairs telomere maintenance.

A. Serial dilutions of the indicated U. maydis strains were directly spotted onto a YPD plate or first subjected to UV irradiation prior to spotting. The plates were incubated for 2 days (-UV) or 3 days (+UV) and the results photographed. B. Serial dilutions of cultures of UCM350 and pot1ΔN that had been passaged for the indicated numbers of streaks were spotted onto a YPD plate and incubated for 2 days. C. Chromosomal DNAs from two independently propagated clonal cultures of UCM350 and pot1ΔN were subjected to TRF Southern analysis at the indicated passages (i.e., Streak 3, 6 and 10). Mean TRF lengths from the pairs of assays were calculated and plotted at the bottom. P-values were calculated using two-tailed Student’s t tests and displayed. D. Chromosomal DNAs from two independently propagated clonal cultures of UCM350 and pot1ΔN strains were subjected to STELA analysis at Streak 10. Mean STELA lengths were calculated from 3 independent assays of each DNA sample and shown at the bottom.

Transcriptional repression of Umpot1 triggers severe growth defects and multiple telomere aberrations

We next interrogated the telomere protection function of U. maydis pot1. Because deleting this gene is likely to be lethal, we generated a conditional mutant (pot1) by replacing the endogenous pot1 promoter with that from crg1, which supports elevated transcription in arabinose-containing media (YPA) but is strongly repressed in glucose-containing media (YPD). To assess the OB-N of Pot1 in telomere protection, we also substituted the pot1ΔN promoter with the crg1 promoter (pot1ΔN). Both mutants exhibited normal growth in YPA, but suffered dramatic growth defects in YPD, consistent with telomere deprotection (Figs 4A and S5A and S5B). Indeed, both mutants became growth arrested in glucose, with pot1ΔN yielding especially small colonies consistent with accelerated arrest. Analysis of cell morphology revealed cells of varying sizes and shapes (S5 Fig), suggesting the arrest can occur in different stages of the cell cycle. This makes pot1-deficient cells distinct from several other telomere protein mutants such as ku70 and trf2-deficient cells that show predominantly elongated morphologies consistent with G2/M arrest [23,27].

Fig 4

pot1 deficiency triggers growth arrest and multiple telomere aberrations.

A. Serial dilutions of the indicated strains were grown on YPA (crg1 promoter on) and YPD (crg1 promoter off) plates for 2 and 3 days, and the results photographed. B. Chromosomal DNAs from three independently generated pot1 and pot1ΔN strains grown in YPA and YPD were subjected to telomere restriction fragment analysis. The overall telomere contents of the samples were quantified and plotted on the right. P-values were calculated using two-tailed Student’s t tests. C. Chromosomal DNAs from three independently generated pot1 and pot1ΔN strains grown in YPA and YPD were subjected to C-circle and G-circle assays. The relative levels of C- and G-circles from the three mutants were quantified and plotted at the bottom. P-values were calculated using two-tailed Student’s t tests. D. Chromosomal DNAs from three independently generated pot1 and pot1ΔN strains grown in YPA and YPD were subjected to in-gel hybridization analysis of ssDNA on both the G- and the C-strand. E. The levels of ssDNA from the in-gel hybridization assays were quantified (N = 3) and plotted. P-values were calculated using two-tailed Student’s t test.

Analysis of telomere repeat DNAs revealed a multiplicity of defects in both the pot1 and pot1ΔN mutants grown in YPD, but with some notable differences (Fig 4). First, while the lengths of telomeres (as determined by TRF Southern analysis) became quite heterogenous in both mutants, the overall telomere content was substantially higher in pot1ΔN (Fig 4B). The heterogeneous telomere fragments of pot1 were detected in the absence of PstI digestion, indicating that they exist as extrachromosomal telomere repeat (ECTR) (S5C Fig). Second, while C-circles (a marker of telomere recombination) were found to be elevated in both mutants, the pot1ΔN mutant also harbored high levels of G-circles (Fig 4C). Third, while pot1 exhibited a modest increase in ssDNA on the C-strand (~10 fold), pot1ΔN showed instead extremely high levels of ssDNA on the G-strand (Fig 4D and 4E). Interestingly, the G-strand and C-strand ssDNA each showed some sensitivity to both 3’-overhang and 5’-overhang nucleases (i.e., Exonuclease I and RecJf) (S5D Fig). This can only be explained by ssDNA on linear ECTRs, which is consistent with the high levels of ECTRs detected in Southern analysis. Together, these observations suggest that loss of pot1 triggers high levels of both telomere recombination and resection, leading to increases in both ds and ss telomere DNA, with much of the DNA in the form of ECTR. In addition, the differences between the telomere aberrations in pot1 and pot1ΔN mutants suggest that even at low levels, the residual Pot1 can still alter aberrant DNA repair reaction at telomeres. For example, if Pot1 OB-N interacts with DNA repair factors such as Rad51 to influence DNA processing, the absence of this domain in pot1ΔN could result in differential generation of ssDNA. In support of residual Pot1 expression, RT-qPCR analysis revealed significant pot1 RNA in the pot1 mutant grown in YPD, at ~1/2 to 1/3 of the normal pot1 RNA level in the parental strain (S5E Fig). In contrast to the drastic increases in abnormal structures associated with telomere recombination and resection, PCR analysis of telomere-telomere fusions revealed only low levels of fusions in the pot1 and pot1ΔN mutants grown either in YPA or YPD (S6 Fig). Hence, non-homologous end joining does not appear to be strongly activated by pot1 deficiency.

Interestingly, even when grown in YPA, pot1ΔN exhibited greater telomere length heterogeneity and a modest increase in C-circle levels (Fig 4A and 4B), suggesting mild telomere de-protection. Because this phenotype was not observed in pot1ΔN, it was presumably due to the over-expression of this truncated allele. While the underlying mechanism is unclear, it is again consistent with the notion that the Pot1ΔN may interact differently with DNA repair factors than Pot1. While Pot1ΔN could retain some interaction with Rad51 through its OB1 and OB2 domains, the overall affinity is likely to be weaker than full length Pot1. There could also be as yet unidentified interactions between Pot1 and other DNA repair factors that are altered by removal of OB-N. High levels of Pot1ΔN may therefore cause an imbalance of DNA repair factors at telomeres to trigger telomere deprotection.

The growth and telomere defects of pot1-deficient U. maydis are greatly suppressed by deletions of rad51 and brh2

Recent studies in mammalian cells suggest that pot1 plays an especially prominent role in suppressing homology-directed repair at telomeres [7,9]. However, this function does not appear to be well conserved in fission yeast [13]. To examine the roles of HDR factors in pot1-deficient U. maydis, we analyzed the phenotypes of the pot1 rad51Δ and pot1 brh2Δ double mutants. Remarkably, both double mutants showed substantially improved growth and manifested more normal morphology in comparison to pot1 when grown in YPD (S7 Fig). Thus, Rad51 and Brh2 were evidently required for the toxicity and growth arrest triggered by pot1 deficiency, i.e., by mediating aberrant repair. Indeed, analysis of telomere repeat DNAs in the double mutants revealed substantial albeit incomplete suppression of the telomere abnormalities observed in pot1 when the strains are grown in YPD (Fig 5). For example, both the abnormally long and short telomere fragments detected in pot1 are greatly reduced (but not eliminated) in pot1 rad51Δ and pot1 brh2Δ (Fig 5A). Similarly, the extremely elevated C-strand ssDNA levels are suppressed by ~10 to 20-fold in the double mutants (Fig 5B). Finally, the levels of C-circles (a marker of telomere recombination) in pot1 are also reduced by more than 10-fold when either rad51 or brh2 is deleted (Fig 5C). Hence, the two core HDR factors appear to play a pivotal role in generating aberrant telomere structures in the absence of pot1. These results are like those observed in pot1-deficient mammalian cells [7], supporting the utility of interrogating the U. maydis mutants in dissecting the underlying mechanisms.

Fig 5

Telomere aberrations in pot1-deficient U. maydis are greatly suppressed by rad51Δ and brh2Δ.

A. Chromosomal DNAs from the indicated wild type and mutant strains grown in YPA and YPD were subjected to telomere restriction fragment analysis. Two independently generated pot1 strains were analyzed in all the assays in this figure. B. Chromosomal DNAs from the indicated wild type and mutant strains grown in YPA and YPD were subjected to in-gel hybridization analysis of ssDNA on both the G- and the C-strand (left). The levels of ssDNA from two independent sets of assays were quantified and plotted (right). P-values were calculated using two-tailed Student’s t tests and displayed. C. Chromosomal DNAs from the indicated wild type and mutant strains grown in YPA and YPD were subjected to C-circle assays (left). The relative levels of C-circle signals from these strains were quantified and plotted (right). Data are from two independent sets of assays. P-values were calculated using two-tailed Student’s t tests and displayed.

It is worth noting that like rad51Δ and consistent with our previous report [24], the brh2Δ single mutant also exhibited significant telomere shortening. Therefore, even though there is no detectable physical interaction between Pot1 and Brh2 (Fig 2A), the latter may nevertheless cooperate with Rad51 to facilitate telomere maintenance, as has been reported for mammalian cells [33]. Our results suggest that this pathway could be modulated by Pot1-rad51 interaction. One other notable result, from the ssDNA analysis, was the frequent detection of prominent ssDNA signals (both G and C-strand) in the wells for strains bearing either rad51Δ or brh2Δ mutations regardless of the pot1 status (Fig 5B, lanes marked with asterisks). This could be due to the accumulation of replication or recombination intermediates, which are known to manifest slow mobility, and is consistent with a role for Rad51 and Brh2 in promoting telomere replication [24].

Upregulation of telomere-repeat containing RNAs is a cardinal feature of pot1-deficient cells

While the observations above implicate Rad51 and Brh2 in promoting aberrant telomere repair in pot1-deficient U. maydis, the mechanisms by which the various telomere aberrations are produced and how they trigger cell cycle arrest remain incompletely understood. In this regard, it is worth noting that both Pot1 and Rad51 have been linked to the regulation of telomere repeat-containing RNA (TERRA), which is proposed to be a critical determinant of telomere maintenance and stability [34]. It should be noted that TERRA generally refers to RNAs synthesized by Pol II from subtelomeric promoters and elongated into the G-strand repeats; most studies to date addressed the roles of this non-coding RNA species at telomeres [35,36]. However, RNAs that carry the telomere C-strand repeat, named ARIA, have also been reported in S. pombe [37,38]. In support of the nexus linking pot1, rad51 and TERRA, POT1 deficiency in human cells was shown to increase TERRA-DNA hybrid formation [7], whereas RAD51 is proposed to directly promote such hybrids through its strand exchange activity [39]. These precedents argue that TERRA may participate in the pathways mediated by pot1-deficiency and rad51/brh2 deletions.

To characterize the potential involvement of telomere-encoded RNA in our mutant strains, we first analyzed the existence of such RNAs by Northern analysis and found little RNA in wild type cells but high levels of transcripts in pot1-deficient mutants. Using strand-specific probes, we showed that RNAs corresponding to both G- and C-strands of telomeres were strongly up-regulated in the pot1 deficient mutant (Fig 6A and 6B). These transcripts were heterogeneous in size but predominantly short (< 1000 nt). RT-PCR analysis confirmed the upregulation of both G- and C-strand RNAs and further suggests that these RNAs include both the subtelomeric region and telomere repeats (S8 Fig). For example, the UT6 subtelomeric region can be amplified using cDNA generated by the CCCTAA4 primer, consistent with the existence of G-strand TERRA that spans the subtelomeric-telomeric junction (S8 Fig). Likewise, heterogeneous PCR products were generated when C-strand RNA was reverse transcribed with a UT6 primer and then amplified using forward and reverse primers that anneal to UT6 and telomere repeats, respectively (S8 Fig). This again implies the existence of C-strand RNAs that span the subtelomeric-telomeric junction. Notably, our data do not exclude the presence of C-strand RNAs that comprise exclusively of just the telomere repeats, called ARIA [38]. However, to avoid confusion, we will refer to our C-strand subtelomere-telomere RNAs as C-TERRA. In addition, our data are consistent with the existence of C-strand RNAs that comprise exclusively of subtelomere sequence, named ARRET (S8 Fig). Altogether, the Northern and RT-PCR analyses demonstrate that abnormally high levels of both G- and C-strand RNAs are present in pot1-deficient cells, due either to increased transcription or to increased RNA stability. Although the end-point PCR assays could not provide quantitative assessment of RNA levels, the relative signals suggest that C-TERRA was present at higher levels in the mutant (see below for additional evidence).

Fig 6

Telomere repeat-containing RNAs are elevated by pot1-deficiency and suppressed by rad51Δ and brh2Δ.

A. RNAs from the indicated wild type and mutant strains grown in YPD were isolated by acid phenol extraction and subjected to Northern analysis using sequentially probes that detect G-strand repeats, C-strand repeats, and 26S rRNA. B. The ratios of G- and C-strand RNA signals to the 26 rRNA signals from Northern analysis were calculated, and then normalized against the ratio of the pot1 mutant and plotted. The normalized values for the pot1 G- and C-strand RNA levels were each set to 100. C. RNAs from the indicated wild type and mutant strains grown in YPD were isolated by acid phenol extraction, treated with Turbo DNase (Thermo Fisher Inc.), and subjected to dot blot analysis using sequentially probes that detect the G-strand repeats, C-strand repeats, and 26S rRNA. D. The ratios of G- and C-strand signals to the 26 rRNA signals from dot blot analysis (n = 2 or 3) were calculated, and then normalized against the ratio of the pot1 mutant and plotted. P-values were calculated using two-tailed Student’s t tests and displayed.

We next compared the levels of telomere repeat RNAs in the pot1 single and double mutants by both Northern and dot blots (Fig 6A–6D). Both analyses showed that while there were drastic increases in TERRA and C-TERRA (by > 20-fold) in the pot1 single mutant, these increases were strongly suppressed by rad51 and brh2 deletions (by > 5-fold). Thus, aberrant transcription of telomere repeats from both G- and C-strands is also regulated by HDR factors in pot1-deficient U. maydis. Lastly, we used specific nuclease treatments to assess the potential existence of RNA-DNA hybrids in the acid phenol-extracted nucleic acid preparations (prior to DNase I treatment), which should be enriched for both RNA and RNA-DNA hybrid (S9 Fig). Interestingly, we found that the G-strand hybridization signals in the pot1, pot1 rad51Δ, and pot1 brh2Δ mutants were all substantially reduced by pre-treatment with DNase I, implying that the G-strand DNA: C-TERRA hybrid (hybrid I) represents a substantial fraction of the signals. The signals were also partially sensitive to RNase H cleavage, indicating the presence of the alternative hybrid (C-strand DNA: TERRA; hybrid II). In contrast, the C-strand signals in the dot blot assays were mostly insensitive to either nuclease, suggesting that they were predominantly in the form of ssRNA or RNA-RNA hybrid. Notably, the C-strand RNA in hybrid I should be degraded by RNase H and should render the C-strand dot blot signal RNase H-sensitive if hybrid I is abundant relative to free C-strand RNAs. This not being the case, we surmise that telomere C-strand RNA may be substantially more abundant than G-strand TERRA in the pot1 mutant, implying differences between these two RNA species with respect to their production or stability.

Discussion

There is growing evidence that HDR factors play multi-faceted and seemingly contradictory roles at telomeres. In the context of normal telomere protection, HDR factors facilitate telomere maintenance by stabilizing stalled forks or promoting repair of truncated telomeres [33]. However, when protection is compromised, HDR factors are often responsible for triggering further aberrations and exacerbating genomic instability [9,10]. How telomere proteins interact physically and functionally with DNA repair factors to achieve context-dependent regulation remains a key area of investigation. In this report, we focus on the interplay between the G-strand binding protein Pot1 and the core HDR factors Rad51 and Brh2/BRCA2 using a fungal model that recapitulates crucial aspects of mammalian telomere regulation. We showed that Pot1 negatively regulates Rad51 and Brh2 to prevent aberrant repair that results in loss of cell viability, but also positively regulates Rad51 activity to promote telomere maintenance, providing an interesting case study for how a single telomere protein can achieve dual regulation of the same DNA repair factor. We also show that up-regulation of aberrant transcription from both G- and C-strand of telomeres is a consistent consequence of telomere deprotection. In addition, we discovered an extra N-terminal OB-fold in the Pot1 orthologs of basidiomycetes, which is absent in other fungal phyla but shared by the majority of metazoans. The mechanistic and evolutionary implications of these observations are discussed below.

U. maydis Pot1 suppresses the toxic activities of core HDR factors at telomeres

Previous analysis of S. pombe Pot1 (SpPot1) suggests that fungal Pot1 may act differently than the mammalian orthologs in telomere protection. To wit, rather than aberrant recombination, loss or inactivation of SpPot1 leads to rapid telomere resection/loss followed by frequent circularization of chromosomes to stabilize the genome and sustain cell viability [12,13]. In contrast, we showed that the phenotypes of UmPot1-deficient cells are very similar to those of comparable mammalian cells, suggesting that suppression of HDR activities may be a widely conserved function of Pot1 in evolution. Importantly, the loss of viability triggered by U. maydis pot1-deficiency is almost completely suppressed by rad51Δ or brh2Δ, confirming the toxicity of HDR factors in the context of telomere dysfunction. This toxicity is at least somewhat specific given that rad51Δ fails to ameliorate the telomere toxicity triggered by ku70 deficiency [23]. Instead, the ku70 deficiency-induced abnormalities are completely suppressed by concurrent blm deletion [25]. Thus, there is a clear division of labor among telomere proteins in managing aberrant repair. The mechanistic underpinnings for this remain unclear and demand further investigation.

Viewed from another perspective, it is striking that in U. maydis, ku70 and pot1 become dispensable for cell viability when specific DNA repair factors are deleted. Such examples of telomere protein dispensability in the context of DNA repair deficiency reify the concept that telomeres have evolved to restrain aberrant repair. It is worth noting that in mammalian cells, there are few examples where the loss of DNA repair factors has been shown to restore the viability of cells missing a key telomere protein. This is probably because mammalian DNA repair factors are often essential for viability in normal mitotic cells. The reduced dependency of U. maydis on DNA repair makes it possible to firmly establish the causal connections between telomere deprotection, aberrant telomere DNA repair, and loss of cell viability.

U. maydis Pot1 promotes telomere maintenance by enhancing the function of Rad51

POT1 has been proposed to promote telomere maintenance in mammalian cells through multiple mechanisms. First, POT1 unwinds G-quadruplexes in vitro, and this biochemical activity is thought to facilitate telomerase activity and telomere replication [40,41]. Second, POT1 binds to and acts in conjunction with the CST complex to facilitate telomere replication and C-strand fill-in synthesis [14,18]. Our analysis of U. maydis Pot1 suggests an additional, non-mutually exclusive mechanism, namely through the regulation of Rad51 activity. Notably, while we showed that Pot1 can stimulate the strand exchange activity of Rad51 through direct protein-protein interaction, how this in vitro regulation reflects the in vivo mechanism of Rad51 at telomeres remains unclear. If Pot1 enhances the formation or stability Rad51 filaments, this could promote complete replication by stabilizing stalled forks. However, it is also clear that rampant strand exchange by Rad51 at telomeres is toxic to the cell and is restrained by Pot1 (see the section above). Thus, it is possible that Pot1-Rad51 interaction may positively regulate some aspects of Rad51 activity while suppressing others in a context dependent manner, e.g., through modulation by other telomere and DNA-repair factors. More studies will be necessary to disentangle the molecular basis of Pot1-mediated regulation of Rad51 in U. maydis.

Notably, mammalian RAD51 is also implicated in telomere replication [33], but a direct POT1-RAD51 connection has not been reported. Instead, there is substantial evidence that RAD51 is regulated by CST, the complex that acts in parallel with shelterin to promote telomere maintenance. For example, CST associates with Rad51, and CST deficiency impairs the recruitment of RAD51 to genomic sites of replication stress [42]. In addition, CTC1 disease mutations that compromise telomere maintenance often disrupt its association with RAD51 [43]. Whether POT1 acts in conjunction or in parallel with CST to regulate RAD51 function at mammalian telomeres is an interesting question for future analysis. Conversely, it will be worthwhile to determine if U. maydis CST subunits regulate Rad51 function. However, it should be noted that only Stn1 and Ten1 homologs are readily discernible in the U. maydis genome (just like S. pombe), suggesting that some fungal lineages may have undergone significant remodeling of the CST complex [24,44].

Aberrant transcription of telomere repeats at de-protected telomeres

There is growing evidence that while TERRA is crucial for normal telomere regulation [34,45], it is often aberrantly expressed from damaged telomeres and can promote abnormal telomere metabolism by forming RNA-DNA hybrids or by interacting with a multiplicity of factors [46-49]. Of more direct relevance to the current study, the depletion or knockout of shelterin components in both mammals and fungi has been shown to trigger the production of telomere repeat RNAs as well as RNA-DNA hybrids. For example, depleting human TRF2 leads to rampant up-regulation of TERRA, which in turn alters histone methylation and promotes end-to-end fusion [50]. Similarly, knocking out human POT1 triggers both abnormal recombination as well as the accumulation of telomere RNA-DNA hybrid [7]. Consistent with these precedents, we observed dramatic upregulation of telomere repeat containing RNAs in pot1-deficient U. maydis. However, both the structure of the RNAs and their relative expression manifest notable features.

One notable feature is the structure of the C-strand RNA. In mammalian cells, this RNA species is evidently expressed at very low levels (if at all) from normal telomeres, and its structure has not been investigated in detail [35,36]. In S. pombe, the C-strand RNAs are synthesized to some degree from normal telomeres and are dramatically up-regulated in a number of mutants, including rap1Δ, taz1Δ, and poz1Δ [38,51]. However, unlike C-TERRA in U. maydis, which encompasses both subtelomere and telomere repeats, the S. pombe C-strand RNAs are reported to comprise just the telomere repeat (ARIA) or subtelomere regions (ARRET). This discrepancy indicates either there are species-specific differences in the biogenesis of C-strand RNA, or the S. pombe ARIA and ARRET could arise through post transcriptional processing. An unresolved conundrum concerning C-strand repeat RNA (ARIA or C-TERRA) is that transcription must initiate within the telomere repeat region, which (unlike subtelomeric Pol II promoters responsible for TERRA transcription) does not contain promoter- or initiator-like elements. Thus, an unusual, promoter-independent mode of RNA synthesis is probably involved. A potential precedent for this is provided by a recent report of MRN-dependent transcription by RNA Polymerase II at DSBs [52]. Regardless of the potential mechanisms, our observation suggests that C-strand RNA synthesis may be relatively common at deprotected telomeres and is worthy of further investigation. Conversely, the growing evidence for promiscuous transcription at de-protected telomeres suggests that a key function of telomere proteins is in fact to suppress aberrant transcription.

Another unexpected finding is the greater abundance of C-TERRA relative to TERRA in the pot1 mutants. As described before, both the RT-PCR and dot blot analyses support the idea that C-TERRA is present at higher levels when pot1 is depleted (S8 and S9 Figs). Whether this is due to enhanced synthesis or greater stability of the C-strand RNA is unclear. The high levels of C-TERRA may partially account for the formation of hybrid I (G-strand DNA: C-TERRA), which was readily detected in the mutant. Most studies of telomere RNA-DNA hybrid have focused on the roles of hybrid II (C-strand DNA: TERRA) and the associated R-loops, and have implicated these structures in telomere abnormalities. Our identification of hybrid I uncovers yet another structure that could be responsible for exacerbating telomere damage.

Implications for the evolution of telomere protein complexes

The discoveries that basidiomycete Pot1 has four OB-fold domains and that UmPot1 interacts with Rad51 have evolutionary implications. Based on structural and functional considerations, it has been argued that RPA, CST and POT1-TPP1 may represent ancient paralogs (i.e., RPA1 ≈ CTC1 ≈ POT1 and RPA2 ≈ STN1 ≈ TPP1) and accordingly may share more structural and functional similarities than hitherto realized [17]. Our analysis, coupled with that by Myler et al. [28], indicates that the common ancestor of fungal and metazoan POT1 probably contained four OB-folds and utilized the two central OBs (OB1 and OB2) for high affinity DNA-binding, just like RPA1. This notion reinforces the growing evidence for structural similarities–and hence evolutionary kinship–between these OB-fold-rich complexes (S10 Fig) [53]. Also, as discussed above, the regulation of RAD51 by CST in mammals and its regulation by Pot1 in U. maydis underscore functional similarities between CST and POT1-TPP1, albeit in different organisms. Further analysis of RPA, CST and POT1-TPP1 in fungi, metazoan and deeper branches of eukaryotes should allow us to adjudicate the evolutionary relationships between these complexes.

Materials and methods

Pot1 homolog identification, alignment, structural prediction, and phylogenetic analysis

Fungal homologs of UmPot1 were identified using BLASTp or PSI-BLAST with appropriate taxonomic restrictions. Similar numbers of homologs that belong to the Basidiomycota, Ascomycota, and Chytridiomycota/Zoopagomycotina lineages were collected for further analysis. The IDs for these homologs are listed in S3 Table. Homologs were aligned using either PSI-Coffee (http://tcoffee.crg.cat/) or PROMALS3D (http://prodata.swmed.edu/promals3d/promals3d.php), and the output displayed using MView (https://www.ebi.ac.uk/Tools/msa/mview/; set to default parameters except coloring by group). The secondary structure predictions associated with the alignments were obtained from the PROMALS3D output and added to the MView displays. Homology modeling was carried out using either PHYRE2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) or SWISS-MODEL (https://swissmodel.expasy.org/) and the predicted structures displayed using PYMOL. To test the hypothesis that basidiomycete OB-N may have arisen through duplication of OB1, we assessed the similarity of basidiomycete OB1 to both basidiomycete OB-N and metazoan OB-N. Briefly, a multiple sequence alignment between the three groups of OB folds was generated using T-Coffee. The Phylip alignment was then used to infer phylogeny via FastME 2.0 (http://www.atgc-montpellier.fr/fastme/) [54] and the results plotted using FigTree [55]. The same strategy was used to analyze basidiomycete Pot1 OB2 and RPA1 OB-N as possible sources of basidiomycete Pot1 OB-N.

Ustilago maydis strain construction and growth conditions

Standard protocols were employed for the genetic manipulation of U. maydis [56-58]. All U. maydis strains used in this study were haploid and were derived from the UCM350 background [57,59] and are listed in S1 Table.

The pot1 strain was constructed by integrating a linearized pRU12-pot1 plasmid containing fragments that correspond to (1) 700 bp of the pot1 promoter and (2) the first 700 bp of the pot1 ORF into the pot1 genomic locus in UCM350. Briefly, the two pot1 fragments were generated by PCR using appropriate oligos with flanking restriction enzyme sites (S2 Table), and then cloned together in between the NdeI and EcoRI sites of pRU12 [60]. Correct recombination of XbaI treated pRU12-pot1 into the genomic locus results in the insertion of the cbx marker and the crg1 promoter precisely adjacent to and upstream of the pot1 ATG initiation codon. Following transformation, the correct mutant strains (pot1) were selected on YPA supplemented with carboxin (4 μg/ml) and confirmed by PCR. The pot1ΔN was generated in identical fashion except that nucleotide (nt) 1126 to 1825 of the pot1 ORF was used in place of the nt 1 to 700 fragment in the cloning of the pRU12-pot1ΔN disruption plasmid. The pot1ΔN strain was constructed by integrating linearized pCbx-pot1ΔN, a derivative of pRU12-pot1ΔN that contains (i) a 5’ homology region with nt -1400 to -700 of the pot1 promoter, and (ii) a 3’ homology region with nt -700 to -1 of the pot1 promoter fused to nt 1226 to 1825 of the ORF. The rad51Δ and brh2Δ disruption cassettes have been described before [24]. The pot1 rad51Δ and pot1 brh2Δ mutants were generated by transforming pot1 with the same rad51 and brh2 knockout cassettes as those used for the derivation of the single mutants [24]. The pot1 and pot1ΔN strains were grown in YPA (1% yeast extract, 2% peptone, 1.5% arabinose) or YPD (1% yeast extract, 2% peptone, 2% dextrose) to induce or repress pot1 expression, respectively.

For growth analysis of the mutants, liquid cultures of U. maydis strains were diluted to OD600 of 0.2 (corresponding to ~ 1.5 x 106 cells/ml). Four microliters of 6-fold serial dilutions of the cultures were then spotted onto YPA or YPD plates and grown at 30°C for 2 to 3 days.

RT-qPCR analysis of pot1 RNA levels

For RT-qPCR analysis of pot1 expressions levels, RNAs were extracted and purified from log-phase U. maydis cultures using acid phenol extraction [61]. The samples were treated with TURBO DNase (Thermo Fisher) and then reverse transcribed in 20 μl reactions that contained 1x SSIV Buffer, 12.5 μM primer (Pot1-PCR-3020R), 1 mM dNTP, 5 mM DTT, 20 U NxGen RNase Inhibitor (Lucigen), and 40 U Superscript IV RT (Thermo Fisher). The reaction mixtures were incubated at 55°C for 40 min and then heated at 70°C for 15 min to inactivate the reverse transcriptase. qPCR was carried out in Chai Open qPCR Dual Channel thermocycler using Chai Green Master mix with 8 μM primers (Pot1-PCR-2821F and Pot1-PCR-3020R) and 1 μl of cDNA. Cycling parameters were as follows: 45 cycles of 95°C for 30 sec, 53°C for 20 sec, 72°C for 40 sec.

Telomere length and structural analyses

Southern analysis of telomere restriction fragments (TRF Southern) was performed using DNA treated with PstI as described previously [24,26]. The blots were hybridized to a cloned restriction fragment containing 82 copies of the TTAGGG repeats. For quantitation, we used the formula ‘mean TRF length = Σ (ODi × Li) / Σ (ODi)’ because the majority of TRF length variation stems from differences in subtelomeric lengths [62,63]. The in-gel hybridization assays for detecting ssDNA on the G- and C-strand have also been described [23]. STELA for telomeres bearing UT4 subtelomeric elements was performed essentially as reported before [22]. To detect telomere-telomere fusions, chromosomal DNA was subjected to PCR using two subtelomeric primers (UT4-F and UT6-F) that are extended by DNA polymerase towards the chromosome ends. The PCR reactions (20 μl) contained 1x Failsafe PreMix H, 0.1 μM each of UT4-F and UT6-F, and 2 U Failsafe polymerase; the thermocycling consisted of 36 cycles of 94°C for 30 sec, 63°C for 30 sec, and 72°C for 2 min. The fusion PCR products were detected by Southern using either a UT4 or UT6 subtelomeric probe [27]. C-circle and G-circles assays were performed as before except that NxGen phi29 DNA Polymerase (Lucigen Corp.) were used for rolling circle amplifications [26].

Analysis of telomere repeat-containing RNAs

RNAs were purified from log-phase U. maydis cultures using acid phenol extraction [61]. For Northern analysis, 3 μg of total RNA were resolved by electrophoresis through a 1.0% agarose/formaldehyde gel [64]. After photography under UV light to visualize ethidium bromide-stained RNAs, the gel contents were transferred to a Hybond-N membrane (GE Healthcare) and hybridization was performed in Church mix using a 32P-labeled single-strand oligonucleotide complementary to the G-strand repeats (CCCTAA8). Following exposure to a phosphor screen, the blot was stripped and first re-probed for the C-strand telomere repeat RNA using 32P-labeled TTAGGG8, and subsequently for 26S rRNA using a PCR fragment (amplified with the UmrRNA_26S_121F and UmrRNA_26S_642R primers (S2 Table)). For dot blot analysis, 0.2 μg of RNAs (untreated or pre-treated with various nucleases) in 10 μl were diluted with 30 μl of RNA incubation/denaturation solution (65.7% formamide, 7.7% formaldehyde, 1.3x MOPS buffer), heated at 65°C for 5 min, and then cooled on ices. After adding 40 μl of 20x SSC, the entire samples were applied to Hybond-N using a Bio-Dot Microfiltration Apparatus (Bio-Rad Laboratories, Inc.). After UV crosslinking, the blot was hybridized sequentially to the same probes as those for Northern analysis. For end-point RT-PCR, the RNA samples from acid phenol extraction were first treated with Turbo DNase (Thermo Fisher Inc.). Fifty ng of RNAs were subjected to reverse transcription using Superscript III RT at 55°C for 40 min, followed by heating at 70°C for 15 min to inactivate RT, and then subjected to PCR (33–37 cycles of 95°C for 15 sec, 66°C for 15 sec, and 72°C for 30 sec) with Q5 polymerase (NEB Inc.) and appropriate primers (S2 Table).

Purification of Pot1-FG and truncation variants

To express full length and deletion mutants of U. maydis Pot1, we amplified the pot1 open reading frame carrying a C-terminal FLAG tag from genomic DNA by PCR amplification using suitable primer pairs (S2 Table). The PCR fragments were cloned into the pSMT3 vector to enable the expression of His6-SUMO-Pot1-FG fusion proteins. BL21 codon plus strains bearing the expression plasmids were grown in LB supplemented with kanamycin and induced by IPTG as previously described [26]. Each fusion protein was purified by (i) Ni-NTA chromatography, (ii) ULP1 cleavage and, and (iii) anti-FLAG affinity chromatography as previously described.

EMSA assays

For DNA binding assays, purified Pot1-FG was incubated with 1–10 nM P32-labeled probe and 0.5 μg/μl BSA in binding buffer (25 mM HEPES-KOH, pH 7.5, 5% glycerol, 3 mM MgCl2, 0.1 mM EDTA, 1 mM DTT) at 22°C for 15 min, and then subjected to electrophoresis at 0.8 V/cm through a 6% non-denaturing polyacrylamide gel in 1x TBE, followed by PhosphorImager analysis.

Pull down assays for Pot1-Rad51 and Pot1-Brh2 interactions

Pot1-FLAG was immobilized on FLAG affinity resin and incubated with 1 μM purified Rad51 or MBP-Brh2/DSS1 complex at 4°C in FLAG(65) buffer (50 mM Tris-HCl, pH 7.5, 65 mM NaCl, 10% glycerol, 0.1% NP-40, 2.5 mM MgCl2, 1 mM DTT). Following incubation with mixing on a rotator at 4°C for 1 h, the beads were washed 4 times with 0.5 ml of the FLAG(100) buffer (same as FLAG(65) except that NaCl is at 100 mM), and then the bound proteins eluted with 60 μl FLAG(150) (same as FLAG(65) except that NaCl is at 150 mM) containing 0.2 mg/ml FLAG3 peptide. The eluates were analyzed by Western Blot using anti-Rad51 or anti-MBP antibodies.

Rad51 strand exchange assays

The assays were modified from published protocols [31,32] and were performed in 15 μl of SE buffer (25 mM Tris.HCl, pH 7.5, 25 mM KCl, 2.4 mM MgCl2, 1 mM DTT, 2 mM ATP) containing 0.25 μM labeled dsDNA (39-mer with the P32-labeled top strand), 0.75 μM unlabeled ssDNA (70-mer complementary to the top strand of the 39-mer), and indicated amounts of Rad51 and Pot1-FG. After 20 min of incubation at 35°C, the reactions were terminated by the addition of 5 μl stop solution (80 mM EDTA, 4% SDS) containing 0.5 μg/μl proteinase K, and incubated for another 20 min at 35°C. Following the addition of 4 μl loading dye (40% glycerol, 0.25% bromophenol blue, 0.25% xylene cyanol), the samples were analyzed by electrophoresis at 0.8 V/cm through a 10% polyacrylamide gel in 1x TBE, and visualized by PhosphorImager scanning.

Comparisons of Pot1 homologs in metazoans and fungi.

A. Multiple sequence alignment of the putative OB-N domains from metazoans and basidiomycetes. The alignment and secondary structure predictions were performed using PROMAL3D and displayed using MView. Amino acids were colored by groups. B. Multiple sequence alignment of the OB3 domains of Pot1 homologs from Basidiomycota, Chytridiomycota, and Zoopagomycota. C. Multiple sequence alignment between the OB3 domains of Pot1 homologs from Basidiomycota and Ascomycota. The putative Zinc-binding residues in the basidiomycete OB3 domains are highlighted.

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Phylogenetic analysis of Pot1 OB-N in relation to (i) OB1 and OB2 in Pot1 and (ii) OB-N in RPA70.

A. To analyze the relatedness of basidiomycete Pot1 OB1 to the OB-N domains from basidiomycete and metazoan Pot1, we generated a multiple sequence alignment between these three groups of OB folds using T-Coffee. The Phylip alignment was used to infer phylogeny via FastME 2.0 (http://www.atgc-montpellier.fr/fastme/) [54] and the result was plotted using FigTree [55]. B. The same analysis was performed for basidiomycete Pot1 OB2 vis-à-vis basidiomycete and metazoan Pot1 OB-N domains. C. The same analysis was performed for fungal RPA1 OB-N vis-à-vis basidiomycete and metazoan Pot1 OB-N domains.

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The Rad51-binding and stimulatory activities of Pot1 truncation variants.

A. The domain structure of Pot1 and the truncation variants analyzed in this study are shown. B. Affinity purified Pot1 proteins were analyzed by SDS-PAGE and Coomassie staining. C. Purified Rad51 was subjected to pull down analysis using FLAG-tagged Pot1 and truncation derivatives. The eluates were analyzed via Western using anti-Rad51 (for Rad51) and anti-FLAG (for Pot1) antibodies. D. The effects of Pot1 truncations on the strand exchange activity of Rad51 were analyzed using oligonucleotide substrates.

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Genetic interaction between pot1ΔN and rad51Δ; effects of hydroxyurea on telomeres.

A. Chromosomal DNAs from three independently propagated cultures of each strain were isolated after ~100 generations of growth (4 streaks) and subjected to telomere restriction fragment analysis. The blot is displayed on the left and the average TRF lengths plotted on the right. B. The UCM350 and pot1ΔN strains were grown in YPD liquid cultures with the indicated concentrations of hydroxyurea and for the specified numbers of days. Chromosomal DNAs were isolated and subjected to telomere restriction fragment analysis.

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Pot1 deficiency impairs cell cycle progression and triggers the production of ECTR.

A. A pot1 liquid culture grown in YPA was harvested, washed 3 times with water, and resuspend in YPD to a starting OD600 of 0.1. The population doubling of the culture was then monitored over a period of 48 hours. B. The UCM350 and pot1 strains were first grown in YPA, and then switched to YPD medium. After another 24 hours of growth, the cells were examined under the microscope. C. Chromosomal DNAs from the indicated strains grown in either YPA or YPD were isolated and subjected to Southern analysis with a telomere repeat probe (TR82) with or without prior PstI digestion. D. DNAs from pot1 and pot1ΔN grown in YPD were treated with the indicated nucleases and then subjected to in-gel hybridization analysis of G- and C-strand ssDNA. The signals were normalized against the untreated sample, and then plotted on the right. E. The pot1 RNA levels from the indicated strains grown in the specified media were analyzed by RT-qPCR. The results were normalized against the signal for UCM grown in YPD, and then plotted.

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Telomere-telomere fusions in pot1-deficient U. maydis.

A. (Top) Schematic diagram of the primers and probes used to detect fusions between UT4- and UT6-containing telomeres. (Bottom) Chromosomal DNAs from the indicated strains grown in either YPA or YPD were subjected to PCR-based fusion detection. Five independent PCR reactions per DNA sample were performed; the products were separated by electrophoresis and subjected to Southern analysis using sequentially a UT4 and a UT6 probe. B. The number of fusion fragments were determined and plotted. Statistical significance was calculated using Students’ test (*, <0.05; **, <0.01; ***, <0.001).

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The growth of pot1, rad51, and brh2 single and double mutants.

A. Serial dilutions of the indicated strains were spotted on YPA and YPD plates. The growth of the strains after 2 and 3 days of incubation were imaged. B. The morphologies of the indicated strains after 24 hours of growth in YPD were examined.

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Pot1 deficiency triggers the accumulation of telomere repeat containing RNAs.

(Left) Schematic illustrations of the structure of UT6 telomeres and the primers used in the RT and PCR reactions designed to detect telomere repeat RNAs. Note that ARRET was previously defined as C-strand RNA comprised of subtelomere sequences only. (Right) RNA samples from the indicated strains grown in YPA or YPD were subjected to RT-PCR using the primers indicated to the left of each panel. Both the F1/R2 and F2/R1 PCR reactions are expected to generate ~200 bp products. The multiple bands detected are likely due to minor insertions/deletions in different copies of UT6 elements [22].

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The nuclease sensitivity of RNA species from telomeres in pot1-deficient U. maydis.

(Top) RNAs from the indicated strains grown in YPD were treated with the indicated nucleases and then subjected to dot blot analyses using sequentially probes for detecting G-strand RNA, C-strand RNA, and 26S rRNA. (Bottom) The ratios of G-strand and C-strand signals to rRNA signals for the untreated, DNase-treated and RNase H-treated samples were calculated. Each ratio was normalized against the untreated sample for the particular strain and plotted.

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Possible evolutionary relationships between three OB-fold rich complexes at telomeres.

The domain structures of RPA, CST and PT subunits are illustrated schematically. The designations for the domains are as follows: OB, oligosaccharide/oligonucleotide-binding; WH, winged-helix; HRJ, Holliday junction resolvase. The OB folds implicated in ssDNA-binding are shaded in dark brown. Available structural and functional evidence strongly suggests that RPA and CST share a common ancestry. Whether PT is derived from the same primordial ssDNA-binding complex is less clear. The demonstration of a 4-OB architecture for Pot1 orthologs in fungi and metazoan suggests that Pot1 and RPA1 have similar domain organizations and may indeed be evolutionarily related.

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U. maydis strains used in this study.

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Oligos used in this study.

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Pot1 homologs utilized for in silico analysis in this study.

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Numerical data for all graphs.

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13 Jan 2022

Dear Dr Lue,

Thank you very much for submitting your Research Article entitled 'Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner' to PLOS Genetics.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This manuscript by Zahid et al. uses both biochemical and genetic approaches to examine the role of the telomeric protein Pot1 in the fungus U. maydis. The data provide an important step forward in understanding the role of this protein at telomeres in U. maydis, a model organism with arguably more telomeric similarities to metazoans than most other fungal model systems. Although most of the results reported bear some similarities to past results in mammalian or other systems, the manuscript should still be of quite broad interest in the telomere field. The manuscript is well written and the results presented are generally of high quality. Some specific criticisms are given below.

P2, l 47-48: This sentence should be altered a bit. I would not say that Rad51 is involved in completing telomere replication as this has not been shown directly here. Length regulation may be perturbed but this is not the same thing.

P3, l 67-70: I suggest modifying the description of telomere erosion. Replication fork blockages by themselves do not shorten telomeres. Presumably, the subsequent processing is doing the actual shortening. Also, substantial erosion will occur via truncations from damage or recombination that are likely independent of replication fork blocks.

It would be useful to mention in the text early on what the sequence and lengths of U. maydis telomeres are.

P5: Some additional information and perhaps analyses should be added regarding the discussion of the additional OB-fold (OB-N). One question is whether five predicted beta folds separated by an alpha helix is adequate as defining an OB-fold. The two predicted structures shown (Fig. 1C) seem rather different from one another and not obviously equivalent (to me at least) to many established beta barrel OB folds and the author’s own evidence indicates that OB-N isn’t involved in DNA binding. Also, where the predicted secondary structural features are should be clearly indicated in Fig. 1B (separately for each of the two species where a predicted structure is shown). Another matter is the question of the origin of the extra OB-fold. The authors favor the idea that it is an ancestral trait, conserved with the N-terminal OB-fold of metazoans. However, the apparent lack of this domain in each of other, more basal groups of fungi certainly disfavors this hypothesis. The authors should compare the sequence of OB-N to that of OB-1 and OB-2. One possibility is that OB-N in basiomycetes represents an independent acquisition of a fourth OB-fold either from an ancient duplication of one of the other OB-folds in Pot1 or perhaps even from some other protein. Comparison of OB-N to OB-1 and OB-2 could help address at least the former possibility. The authors should also discuss a bit more their search comparisons to other fungi. Have all available fungi in Ascomycota, Zoopagomycota and Chytridiomycota been examined? Finding even a single non-basiomycete with the OB-N could greatly strengthen the possibility of multiple independent loss events of OB-N in these other fungal groups. The authors might also want to mention the possibility that an independent acquisition of an OB-N in basiomycetes could conceivably be accompanied by some degree of convergent evolution that might lead to some resemblance to the metazoan OB-N.

P5-6: Some details of the strand-exchange assay should be mentioned in the text. Minimally, the authors should describe whether the assay shown involves telomeric or non-telomeric sequences and what the sequences of the oligos used was. I don’t know if there are technical problems with examining strand exchange with repetitive sequences but doing the reaction with telomeric and non-telomeric repeats would be very interesting. Oligos with either the ss or the ds region telomeric could be used. One might guess that Pot1 would be better at causing strand exchange with telomeric compared to non-telomeric sequences, especially since the reported effect in the data here is quite modest. Strand exchange that favored telomeric sequence would be both especially interesting and harder for skeptics to ignore.

Define the asterisks in the legend to Fig. 2D.

The authors should discuss the additional bands seen in the strand exchange assay, most notably in Fig. 2D and what evidence proves the particular band expected to be the result of strand exchange is actually that. An arrow pointing to this specific band would be helpful too. At least one of the other bands varies in intensity quite considerably, increasing when Pot1 is present.

P6: The variable telomere length phenotype of U. maydis wild type cells complicates trying to prove modest average length changes but the data presented seem adequate for arguing that the Pot1 mutation and the Rad51 mutation do indeed have somewhat shorter telomeres than wild type. The authors argue that the physical interactions of Pot1 and Rad51 and the apparently similar telomere shortening in pot1 delta N and rad51 mutants suggests that these proteins act in the same telomere maintenance pathway. However, the authors’ data in Fig. 5 suggests that brh2 delta mutants also may have similarly shortened telomeres. This ought to be further examined. If true, this would add more information to help elucidate the connection between HR and telomeres in U. maydis.

P7/Fig. 4B: It would be worthwhile to have quantification of the relative telomere signal in the mutants. While this would be subject to some issues regarding interpretation, it would still be interesting to estimate.

Fig. 5C: G circle and C circle data are stated as being shown but only C circle data is shown.

Fig. 6B legend appears to refer to Fig. 6D.

Reviewer #2: The review is uploaded as an attachment.

Reviewer #3: In this manuscript, Dr. Lue and his colleagues identified an extra N-terminal OB fold (OB-N) in fungal Ustilago maydis Pot1 (UmPot1). By sequence alignment, the authors showed that this OB-N is present in most metazoans. Using in vitro biochemical studies, the authors further demonstrated that UmPot1 OB-N, together with OB1 and OB2, binds directly to Rad51 and regulates its strand exchange activity. Deletion of OB-N domain alone resulted in telomere shortening, while deletion of Pot1 through transcriptional repression resulted in growth arrest and severe telomeric structural aberrations as well as high level of TERRA. In addition, the phenotypes can be largely suppressed by co-deletion of rad51 and brh2. Thus, the authors conclude that UmPot1 maintains telomere integrity under both normal and deprotected states through regulation of two major HDR repair proteins Rad51 and Brh2. Overall, the experiments were well designed and carefully characterized. The data is of high quality and presented in a clear and convincing manner. I have a few comments listed below:

1. The authors mentioned evolutionary relationships of RPA like complexes at telomeres. It would be interesting to check the sequence/2D/3D structural similarity between OB-N domain in UmPot1 and the OB fold in RPA1 or CTC1 to get an idea whether UmPot1 is a “chimeric” RPA-Pot1 form.

2. In Figure 3, TRF Southern blot showed Pot1∆N strain had clear telomere shortening, while the deletion seems not affecting overall growth by serial dilution. Have the authors followed a long-term growth effect of Pot1∆N strain? Or in the presence of replication stress regents like HU to see whether it accelerates telomere shortening and senescence?

3. The deletion of Pot1 (Pot1crg1 and Pot1∆Ncrg1) resulted in a substantial increase of ssDNA shown in Figure 4. Are those ssDNA generated by aberrant HDR or by resection, or are those ssDNA located internally or terminally? Is it possible to distinguish it by adding the Exo I digestion?

4. The OB-N is present in most basidiomycete, but not in fission yeast and other filamentous fungi. Have the authors considered generating a chimeric fusion Pot1 with the OB-N from UmPot1 fused with other fission yeast or filamentous fungi Pot1?

Minor points:

1. In Figure 1D, it will be easier for the readers to have the scheme of the truncation variants as shown in S2 figure a.

2. From the biochemical binding experiment, have the authors calculate the stoichiometry ratio of Pot1 and Rad51?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Submitted filename: Critiques.pdf

Click here for additional data file.

15 Mar 2022

Submitted filename: Response to Reviewers comments.pdf

Click here for additional data file.

2 Apr 2022

Dear Dr Lue,

We are pleased to inform you that your manuscript entitled "Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have addressed all my criticisms and I have no further criticisms.

Reviewer #2: Zahid et al. has now revised the manuscript with additional data shown in Figures 1 - 3, S2, S4, and S5. The authors have also revised the text to address the reviewers critiques. I do not have any further concerns about this manuscript.

Reviewer #3: In this revised manuscript, the authors have addressed majority of my questions. I am particularly impressed by the new S5 Figure D that showed both resection and resolution of HDR involved in generation of ssDNA. The overall manuscript is much improved. Given the importance of the topic, it will appeal to a wide audience in the telomere field.

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: No

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22 Apr 2022

PGENETICS-D-21-01605R1

Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner

Dear Dr Lue,

We are pleased to inform you that your manuscript entitled "Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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