Mapping the position of the Pif1 NLS

The cNLS Mapper program was used to search for candidate nuclear localization signals in the Pif1 open reading frame [23]. The program assigns prediction scores of up to 10 with higher scores indicating a higher probability of being an NLS. cNLS Mapper identified two candidate NLSs within Pif1: a monopartite NLS (⁷⁷⁷DEQVKKRKLDY⁷⁸⁷; prediction score of 8) and a bipartite NLS (⁴¹⁷RQRGDVKFIDMLNRMRLGNIDDETEREFKKLSRP⁴⁵⁰; prediction score of 5). The bipartite NLS had a relatively low prediction score, and its sequence overlapped one of the canonical helicase motifs (motif IV) as well as another conserved region (motif A) (Fig 1A). Based on these findings, we focused our analysis on the monopartite NLS candidate sequence.

Strains to test whether putative NLS is required for nuclear functions

To determine if the predicted monopartite NLS (⁷⁷⁷DEQVKKRKLDY⁷⁸⁷) was important for Pif1 nuclear functions, we deleted the core of this sequence, ⁷⁸¹KKRK⁷⁸⁴ to generate the pif1-NLSΔ (NLS deleted) allele (Fig 1B). Only the core of the putative NLS was deleted to reduce the possibility of disrupting sequences important for other Pif1 functions. In addition, these four amino acids match the consensus for a classical monopartite NLS motif: K-(K/R)-X-(K/R) [24].

As a control, we fused the NLS from the simian virus 40 (SV40) T antigen (¹²⁶PKKKRKV¹³²), which functions as an NLS in S. cerevisiae [25], to the end of the pif1-NLSΔ ORF to generate the pif1-NLSΔ+SV40 allele (Fig 1B). If our hypothesis is correct, pif1-NLSΔ cells should be defective in nuclear Pif1 functions, while still retaining mitochondrial activities. In addition, nuclear defects in pif1-NLSΔ cells should be reduced in pif1-NLSΔ+SV40 cells, even though the SV40 NLS is not in the same position as the predicted Pif1 NLS. We also generated a double mutant of Pif1, pif1-m2+NLSΔ, which deleted the candidate NLS from the partial loss of function pif1-m2 allele.

Amino acids ⁷⁸¹KKRK⁷⁸⁴ are not essential for protein stability or for maintenance of mitochondrial function

Western blot analysis showed that the Pif1-NLSΔ protein was stably expressed (Fig 1C). In addition, attaching the SV40 NLS to the carboxyl-terminus of either WT Pif1 or Pif1-NLSΔ did not affect the abundance of either protein (Fig 1C).

If ⁷⁸¹KKRK⁷⁸⁴ is the core of a functional NLS, cells expressing pif1-NLSΔ should have impaired nuclear functions but normal mitochondrial activity. Yeast requires functioning mitochondria to grow on glycerol media. As expected, pif1-NLSΔ and pif1-NLSΔ+SV40 cells grew as well as WT cells on media containing glycerol as the sole carbon source (Fig 2). This finding indicates that deletion of ⁷⁸¹KKRK⁷⁸⁴ did not affect Pif1 mitochondrial function.

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Fig 2. The Pif1 NLS is not required for its mitochondrial function(s).

Ten-fold serial dilutions of pif1Δ cells carrying empty plasmid or plasmids with the indicated PIF1 allele were spotted on plates containing glucose (left) or glycerol (right). The genomic copy of PIF1 was replaced with NatMX.

https://doi.org/10.1371/journal.pgen.1010853.g002

Amino acids ⁷⁸¹KKRK⁷⁸⁴ of Pif1 are essential for suppressing telomere lengthening

To determine if ⁷⁸¹KKRK⁷⁸⁴ is a functional Pif1 NLS, we examined three nuclear phenotypes: telomere length, BIR, and Okazaki fragment processing in pif1-NLSΔ and pif1-NLSΔ+SV40 cells as well as Pif1 binding to specific sites in nuclear and mitochondrial DNA. Because Pif1 displaces telomerase from telomeres [26], pif1Δ cells have long telomeres [4]. Consistent with a lack of nuclear Pif1, pif1-NLSΔ cells had long telomeres that were similar in length to those in pif1Δ cells (Fig 3). This long telomere phenotype was suppressed in pif1-NLSΔ+SV40 cells. The ability of the SV40 NLS to suppress the telomere defects of pif1-NLSΔ cells supports our hypothesis that ⁷⁸¹KKRK⁷⁸⁴ is the core of a functional NLS.

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Fig 3. The Pif1 NLS region is required to maintain wild type telomere length.

Analysis of telomere lengths in two independent isolates of cells expressing PIF1 (WT) and various pif1 mutants. Isolates were streaked at least six times to allow the full impact of each gene on telomere length. Genomic DNA was digested with XhoI and analyzed by Southern hybridization using a probe specific to the Y’ sub-telomeric region.

https://doi.org/10.1371/journal.pgen.1010853.g003

Pif1’s role in break-induced replication (BIR) requires amino acids ⁷⁸¹KKRK⁷⁸⁴

Pif1 is critical for break-induced replication (BIR) [11,12]. Its proposed roles during BIR include unwinding the DNA duplex to allow the progression of BIR, unwinding the D-loop formed by the newly synthesized DNA, and stabilizing Polδ to enhance BIR processivity [12]. To test the effects of deleting ⁷⁸¹KKRK⁷⁸⁴ on BIR [12,27,28], BIR efficiency was determined in derivatives of AM1003 [27], a strain that is well established for BIR assays (Fig 4A). AM1003 and its derivatives are otherwise haploid cells that are disomic for chromosome III and contain a galactose-inducible copy of the HO endonuclease (Fig 4A and S1 Table). One copy of chromosome III, the donor chromosome, is full-length but has a mutant HO recognition site, the MATα-inc allele, that is not cleaved by the HO endonuclease. The other copy of chromosome III (recipient) is truncated at the Z-region of MATa but contains an intact Ya-region and HO recognition site [27]. When galactose is present and HO is expressed, a DSB is induced at MATa on the truncated chromosome III. This DSB is repaired predominantly by BIR that is initiated by strand invasion into the full-length copy of chromosome III (donor). In this system, BIR copying must proceed for ~100 kb to produce a complete BIR outcome, which generates Ade⁺ Leu^- cells (Fig 4A). This system also allows the identification of failed BIR events. In particular, failed DSB repair often leads to the loss of the truncated chromosome and formation of Ade^-red Leu^- colonies (chromosome loss, CL). Another phenotype of failed BIR is Ade^-white Leu^- which is indicative of half-crossover (HC). HCs result from interruption of BIR synthesis, which leads to the fusion between parts of the recipient and donor chromosomes and to the loss of other parts (Fig 4A). In WT (PIF1), the majority of DSBs (~ 75%) were repaired by BIR as monitored by the formation of Ade⁺Leu^- colonies, while failed BIR events were less frequent (~9% of CL and ~4% of HC) (Fig 4B and S2 Table).

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Fig 4. The effects of pif1-NLSΔ on long-range BIR.

(A) Schematic of the BIR genetic assay in a yeast disomic system (AM1003). Repair outcomes are distinguished by genetic markers. (B) The distribution of repair events after DSB induction in WT and indicated mutant strains. Asterisks represent statistically significant difference from WT (PIF1) as determined by contingency test (****: p<0.0001; **: p = 0.0016). (C) The fractions of GCR and no-GCR events among Ade⁺Leu^- repair outcomes as determined by CHEF gel electrophoresis analysis. Asterisks indicates significant differences from WT and N/A = not applicable; NS = not significant (D) The distribution of all repair DSB events, including calculated % of three classes contributing to the formation of Ade⁺Leu^- outcomes (from B), including BIR, half-crossover II (HC-II), and GCRs. The amount of GCRs was calculated by applying the fraction of GCR calculated from C to the total Ade⁺Leu^- from B. The number of HC-II was assumed to be equal to HC-I based on B [27]. (E) An illustration demonstrating the formation of half-crossover events followed by two scenarios of chromatid segregation at mitosis leading to the formation of two half-crossover classes (HC-I and HC-II). In the scenarios shown, only one broken chromatid was repaired while the other was lost. Labels are provided for sister chromatids of both chromosome III homologs (1A, 1B, 2A, and 2B). See Materials and Methods and [27] for details.

https://doi.org/10.1371/journal.pgen.1010853.g004

As shown previously [12,28], the fraction of Ade⁺Leu^- outcomes in pif1Δ cells, was significantly reduced (Fig 4B and S2 Table) indicative of defective BIR. Meanwhile, DSB repair classes representing failed BIR (HC and CL), comprised ~40% of all cases, which was significantly higher than in WT cells (p<0.0001).

Analyses of DSB repair in the pif1-NLSΔ mutant showed that the distribution of its DSB repair outcomes was similar to that of pif1Δ cells (Fig 4B and S2 Table). In particular, the fraction of Ade⁺Leu^- cases was ~45% (significantly lower than in PIF1 (WT); p < 0.0001), while the fraction of failed BIR events (HC, and CL) was ~45%, significantly higher than in PIF1 (WT) (p < 0.0001). Importantly, addition of the SV40-NLS largely compensated for the BIR defects in pif1-NLSΔ cells (Fig 4B and S2 Table). These data show that ⁷⁸¹KKRK⁷⁸⁴ is needed for efficient BIR completion, consistent with the hypothesis that it is the core of a functional NLS. In addition, another separation of function pif1-m2 mutation and the mutant containing two mutations pif1-m2 and pif1-NLSΔ were as defective in completion of BIR as pif1Δ and pif1-NLSΔ (Fig 4B and S2 Table).

BIR in pif1-NLSΔ leads to chromosomal rearrangements

We previously demonstrated that while the majority of Ade⁺Leu^- colonies in WT cells result from completed BIR events, Ade⁺Leu^- colonies in pif1Δ cells frequently represent gross-chromosomal rearrangements (GCRs) [28]. To determine the fraction of GCRs among Ade⁺Leu^- colonies, we used contour-clamped homogeneous electric field (CHEF) gel electrophoresis (similar to described in [27]). As expected, we observed a significantly higher fraction of GCR events among Ade⁺Leu^- colonies in pif1Δ cells (10/23, 43%) as compared to WT cells (1/30, 3%) (see Fig 4C for p-values). Likewise, ~36% of Ade⁺Leu^- events were due to GCR events in pif1-m2 cells. Similar to pif1Δ, ~33% and ~31% of the Ade⁺Leu^- colonies were due to GCR events in pif1-NLSΔ and pif1-m2+NLSΔ cells respectively, again, significantly higher than in WT (Fig 4C). In contrast, the frequency of GCR events in pif1-NLSΔ+SV40 cells was comparable to that of WT cells (Fig 4C).

Moreover, we previously proposed [27] that many of even non-rearranged Ade⁺Leu^- colonies in pif1 mutants likely resulted not from completed BIR events, but from the second type of half-crossovers that we call HC-II (see Fig 4D and schematics in Fig 4E for explanation). In brief, HC-II events result from mitotic segregation of half-crossover chromosomes with an intact copy of the donor chromosome, which leads to the formation of Ade⁺Leu^- colonies. Assuming that the frequencies of Ade^-white Leu^- half-crossover (HC-I) and of HC-II classes are equal [27], we can estimate the fraction of HC-II (invisible) class among Ade⁺Leu^- events for each strain background. Following the subtraction of GCR and HC-II from Ade⁺Leu^- events, it appears that real BIR events are rarely completed in all pif1 mutants including pif1-NLSΔ, but successfully completed in pif1-NLSΔ+SV40 (similarly to WT) (Fig 4D).

Together, we conclude that the pif1-NLSΔ and pif1Δ cells are similarly defective in long range BIR, which requires extensive DNA synthesis (up to 100kb) and which is detected by the BIR assay used here.

pif1-NLSΔ disrupts BIR-associated mutagenesis

To determine if pif1-NLSΔ cells are also defective at earlier stages of BIR, we monitored BIR progression within the first 16kb away from the double strand break by measuring the frequency of BIR-associated mutagenesis [28]. For these experiments, we used a lys2::A₄ reporter gene inserted 16kb centromere-distal to MATα-inc. In this system (Fig 5A), Lys⁺ cells can be generated by frameshift mutations produced within the reporter gene, which are highly stimulated during BIR DNA synthesis [28,29]. Unlike the long-range BIR assay (Fig 4A), a positive signal in the mutagenesis assay (Fig 5A) requires that BIR proceed for only 16 kb.

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Fig 5. The effects of pif1-NLSΔ on BIR-associated frameshift mutagenesis.

(A) Schematic of BIR-associated mutagenesis assay. The rate of mutagenesis was measured by the frequency of Lys^- to Lys⁺ reversion using a lys2::A₄ reporter: A -1bp frameshift within the lys2::A4 reporter generates a Lys⁺ phenotype. The reporter is inserted 16kb centromere distal to MATα-inc on the donor chromosome. Thus, BIR events that proceed at least 16 kb can generate Lys⁺ cells (I) and results in high rate of Lys⁺. When BIR does not reach 16kb, it cannot generate Lys⁺ cells, and the rate of Lys⁺ is low (II). (B) The rate of Lys⁺ events measured 7hrs after a galactose-induced DSB in cells expressing different PIF1 alleles. Asterisks (*) indicates statistically significant differences from WT (PIF1), determined by the Mann-Whitney test (****: p<0.0001; **: p = 0.0089). See also S3 and S4 Tables for the rates of Lys⁺ reversion prior to DSB and for the details of statistical analysis.

https://doi.org/10.1371/journal.pgen.1010853.g005

As in earlier studies [28,29], BIR was associated with a high rate, 3.9x10^-6 of Lys⁺ events in WT (PIF1) cells (Fig 5B and S3 and S4 Tables), and this high rate was galactose dependent (compare 7h versus 0h in S3 and S4 Tables). In contrast, pif1Δ cells had a 27x times lower BIR-associated Lys⁺ mutagenesis than PIF1 (WT) cells (See S3 and S4 Tables) [29]. Thus, in the absence of Pif1, BIR reaches the 16kb position in only ~4% of the events. Consistent with earlier observations [28], BIR-associated frameshifts at 16kb were 5x more frequent in pif1-m2 than in pif1Δ cells, and this level was 5x lower than in WT (Fig 5B and S3 and S4 Tables), confirming previous data that pif1-m2 is not a null allele in this assay [8,28,30].

The level of BIR-associated mutagenesis in pif1-NLSΔ cells was 2.3x lower than in WT (p<0.0001), and 2.2x higher than in pif1-m2 cells (p<0.0001) (Fig 5B and S3 and S4 Tables). Thus, like pif1-m2, pif1-NLSΔ was defective but not a null in the mutagenesis assay. Moreover, pif1-NLSΔ cells were not as deficient as pif1-m2 cells. In addition, the pif1-m2+NLSΔ double mutant was significantly more defective as compared to either single mutant (Fig 5B and S3 and S4 Tables). Nonetheless, even this double mutant was 3.6x less defective than pif1Δ cells (p< 0.0001 for all comparisons). Thus, there must be residual nuclear Pif1 even in pif1-m2+NLSΔ cells. Finally, the level of mutagenesis was restored in pif1-NLSΔ+SV40 compared to pif1-NLSΔ (p<0.0001), but not to the wild type level (p = 0.0089) (Fig 5B and S3 and S4 Tables).

Together, we conclude that all four pif1 mutants, including pif1-NLSΔ, were highly BIR defective as seen by both long- and short-range BIR assays. However, results from the short-range BIR assay indicated that the three pif1 mutants are not equally defective for BIR.

BIR-associated DNA synthesis is defective in pif1-NLSΔ cells

Although both the long- and short-range BIR assays revealed that pif1-NLSΔ cells are BIR-defective, neither assay measures BIR synthesis directly. Therefore, we used the digital droplet PCR (ddPCR) based AMBER (Assay for monitoring BIR elongation rate) assay, which precisely measures the amount of DNA synthesized during individual BIR events [31,32] by monitoring copy number changes at different BIR positions and at multiple time points after galactose addition (Fig 6A, see legend for details). AMBER uses donor-specific primer sets for three separate locations within a 61 kb BIR region and then normalizes these values to the signals obtained at the ACT1 locus to determine relative copy number. Primer sets were located at 1.9kb (P2), 22.4kb (P3), and 61.4kb (P4) centromere distal to the DSB. Copy numbers at these positions are 1x before BIR and can increase to a maximum of 2x following BIR. Evidence of DNA synthesis was defined as an increase in donor DNA copy number of at least 1.1x. At 10hrs after DSB induction, WT PIF1 cells had a 1.7x copy number at P2 and P3 and 1.6x at P4 (See Fig 6B and 6C and S5 Table). In contrast, in pif1Δ cells, at 10 hrs, increased copy number was detected only at P2 and was only ~ 1.1x (Fig 6B and 6C and S5 Table), results similar to an earlier report [32]. In pif1-m2 cells at 10 hrs, a small increase was observed at both P2 and P3 (~1.1x; Fig 6B and 6C), but not at P4. At 10 hrs, pif1-NLSΔ cells had increased copy number at all three sites (~1.3x at P2 and ~1.1x at P3 and P4; Fig 6B and 6C). Since DNA synthesis in Pif1-defective mutants is often incomplete and therefore some newly synthesized DNA may degrade by 10 hours, we also compared the maximum copy number increases reached in different strains between 0 and 10 hrs (see Fig 6D) and came to the same conclusions. In addition, as expected, the copy number was higher in pif1-NLSΔ+SV40 (~1.5x at P2 and ~1.4 at P3 and P4) than in pif1-NLSΔ cells. The extent of BIR synthesis was similar in pif1-m2+NLSΔ and pif1Δ cells (~1.1x at P2; no increase detected at P3 and P4) (Fig 6B, 6C and 6D).

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Fig 6. The effects of pif1-NLSΔ on the extent of BIR synthesis detected by AMBER.

(A) Schematic of AMBER analysis measuring the extent of BIR synthesis in a yeast disomic system (derivatives of AM1003). Colored triangles indicate locations of primer sets. (P1 is located at the left arm of chromosome III (~153kb away from MATα-inc) and used as a control. P2, P3, and P4 are placed 1.9kb, 22.4kb, and 61.4kb centromere distal from MATα-inc. (B) DNA synthesis measured by AMBER in WT (PIF1) and in strains containing various mutations of PIF1 during the first 10hrs following addition of galactose. The results are presented as Boltzmann sigmoidal curve. One out of three independent biological repeats that showed similar results is shown in each panel. (See S5 Table for other repeats). Mean values of target to reference (ACT1) loci ratios were calculated by Poisson distribution based on 20,000 droplets with error bars representing upper and lower Poisson 95% CI. (C, D) The comparison between the extents of BIR synthesis in various pif1 mutants at 10hrs after DSB induction (C) or the maximum detected between 0h and 10hrs (D) as assessed by AMBER. Mean±s.d (n = 3, three independent biological repeats). Note: some values are higher in D than in C due to BIR interruption prior to 10h followed by degradation of newly synthesized DNA in pif1 defective strains.

https://doi.org/10.1371/journal.pgen.1010853.g006

In conclusion, cells lacking ⁷⁸¹KKRK⁷⁸⁴ were defective in BIR initiation and progression and this defective phenotype was largely suppressed in pif1-NLSΔ+SV40 cells. In agreement with the results of BIR-associated mutagenesis assay, these results showed a hierarchy in the BIR defects of the different pif1 mutants: pif1Δ ≈ pif1-m2+NLSΔ > pif1-m2> pif1-NLSΔ>pif1-NLSΔ+SV40>WT.

⁷⁸¹KKRK⁷⁸⁴ is important for Pif1 enrichment at nuclear, but not mitochondrial DNA binding sites

The defects in three different nuclear functions suggests that the nuclear abundance of the Pif1-NLSΔ protein was lower than in PIF1 cells but not as low as in pif1Δ cells. We used chromatin immunoprecipitation (ChIP) of Pif1 to its binding sites as a proxy for Pif1 abundance. For these experiments, PIF1 (WT) and three pif1 mutant alleles were tagged at their C-termini with 13 MYC epitopes [33]. By western blot, all tagged PIF1 alleles were stably expressed (Fig 7A).

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Fig 7. Diminished enrichment of Pif1 protein lacking ⁷⁸¹KKRK⁷⁸⁴ in the nucleus.

(A) Westerns blot analysis of Myc-tagged PIF1 (WT) and pif1 mutant alleles using an anti-MYC or anti-PGK1 (loading control) antibody. (B) Pif1 enrichment measured by ChIP in WT (PIF1) and indicated pif1 mutants at two different nuclear positions (MAT (BIR-specific) and CEN13) and at mitochondrial-specific site (see Materials and Methods for details). Asterisks represent statistically significant difference as determined by unpaired t-test (***: p = 0.0005, *:p<0.05).

https://doi.org/10.1371/journal.pgen.1010853.g007

We analyzed the recruitment of Pif1 during BIR at MAT at 4hrs after DSB induction (see Materials and Methods and [12] for details). As expected, we observed a high enrichment of Pif1 at MAT, while Pif1-NLSΔ MAT binding level was ~4.6x lower than that for WT Pif1 (Fig 7B). We also measured Pif1 binding in cycling cells (no DSB induction) at CEN13, a known site of high Pif1 binding [15]. Again, Pif1-NLSΔ binding to CEN13 was ~4.9x lower than WT Pif1.

As predicted, defective nuclear Pif1-NLSΔ binding was restored in pif1-NLSΔ+SV40 cells at both MAT and CEN13 sites. As expected, Pif1 binding to a mitochondrial location was indistinguishable in PIF1 (WT) and three pif1 mutant cells (pif1-NLSΔ, pif1-m2+ NLSΔ, and pif1-NLSΔ+SV40) (Fig 7B). Together, ChIP data support the hypothesis that pif1-NLSΔ leads to a decrease of Pif1 localization to the nucleus but does not affect its presence in mitochondria.

Partial suppression of dna2Δ lethality by pif1-NLSΔ

PIF1 deletion rescues the lethality of dna2Δ cells [6]. This rescue is thought to reflect the role of Pif1 in Okazaki fragment maturation. The current model suggests that Pif1 and DNA Polymerase δ together generate long Okazaki flaps that cannot be cleaved by flap endonuclease 1 (Fen1). Rather, the long flaps are cleaved by Dna2, an essential multi-functional helicase-nuclease [34,35]. According to this model, if Pif1 is absent, long flaps are not generated, and Dna2 is no longer essential. For Pif1 to promote formation of long flap Okazaki fragments, Pif1 must be nuclear-localized.

To test whether pif1-NLSΔ rescued the lethality of dna2Δ, we first used a qualitative assay where colony formation of pif1Δ dna2Δ cells transformed with centromeric plasmids containing various pif1 alleles was measured. As expected, dna2Δ cells carrying either the PIF1(WT) vector or the vector with pif1-NLSΔ+SV40 were not viable. In contrast, empty vector alone or vector carrying pif1-NLSΔ suppressed the lethality of dna2Δ (Fig 8A; compare NLSΔ (bottom) to “Empty vector” (top)).

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Fig 8. pif1-NLSΔ can partially suppress lethality of dna2Δ.

(A) Schematic of experimental system for the qualitative suppression test is shown on the left. Right: representative samples of viability of pif1Δ dna2Δ cells carrying empty plasmid or plasmids with the indicated PIF1 allele. (B, C) The number and phenotypes of viable spores (colonies) obtained following meiosis of two diploids: DNA2/dna2::URA3 PIF1/pif1::KANMX and DNA2/dna2::URA3 pif1-NLSΔ /pif1::KANMX. The meiotic outcomes were analyzed by tetrad analysis (B) and by random spore analysis (C). G418^s = sensitive to G418 (KANMX absent); G418^r = resistant to G418 (KANMX present).

https://doi.org/10.1371/journal.pgen.1010853.g008

To investigate suppression of dna2Δ in a more quantitative assay, we created two diploid strains that were both DNA2/dna2::URA3 heterozygotes. One of these strains was also PIF1/pif1::KANMX, while the other was pif1-NLSΔ/pif1::KANMX. Both diploid strains were sporulated and dissected. As expected, in the strain heterozygous for PIF1 (WT), there were no viable Ura⁺ G418^S (dna2ΔPIF1) spores (out of 36 dissected tetrads; Fig 8B). Dissection of 35 tetrads from the pif1-NLSΔ/pif1::KANMX strain produced three viable Ura⁺G418^S (dna2Δpif1-NLSΔ) spores while 24 Ura⁺ spores were G418^R (dna2Δ pif1Δ). This result is indicative of a partial suppression of dna2Δ lethality by pif1-NLSΔ. As expected, in both strains, an equal number of G418^R and G418^S was observed among Ura^- (DNA2 wt) colonies (Fig 8B). Our conclusion was further supported by the results of random spore analysis in the same diploids where among 42 Ura⁺ clones generated after sporulation of the pif1-NLSΔ/pif1::KANMX diploid, eight were G418^S (dna2Δpif1-NLSΔ) while 34 were G418^R (dna2Δpif1Δ) (Fig 8C). As expected, no G418^S Ura⁺ (dna2ΔPIF1) colonies were observed in the strain heterozygous for PIF1 (WT) (Fig 8C). Therefore, we conclude that the deletion of PIF1 ⁷⁸¹KKRK⁷⁸⁴ partially suppresses the lethality of dna2Δ cells.

The pif1-NLSΔ allele is not a null for nuclear functions of Pif1

pif1-NLSΔ cells were defective in all tested nuclear functions. In two of these assays, telomere length regulation and long-range BIR, pif1-NLSΔ and pif1Δ cells were similarly defective (Fig 3 and 4). However, in other more sensitive assays, pif1-NLSΔ cells was not a null allele. For example, the very sensitive mutagenesis (Fig 5) and AMBER (Fig 6) assays revealed that both pif1-NLSΔ and pif1-m2 were more active than pif1Δ, consistent with earlier findings that pif1-m2 is not a null allele [8,28,30]. Specifically, the mutagenesis and AMBER assays showed that, in some cells, BIR proceeded for up to 22kb in both pif1-NLSΔ and pif1-m2 cells. In addition, using the AMBER assay, BIR was detected as far as 62 kb from the DSB in pif1-NLSΔ cells, but not in pif1-m2 cells (Fig 6). Even the double mutant pif1-m2+NLSΔ had residual BIR activity in the mutagenesis (but not the AMBER) assay. It is not surprising that the mutagenesis and AMBER assays did not show identical dependencies: the mutagenesis assay detects all completed DSB repair events with BIR synthesis within 16 kb of the DSB, even those that occur with a long delay, as long as they generate viable cells [28]. In contrast, the AMBER assay detects BIR synthesis only during the first 8–10 hours after the DSB [31,32]. On the other hand, the AMBER assay is a direct assay for BIR synthesis and detects not only finished, but also incomplete events [32] while the mutagenesis assay is indirect.

As with the mutagenesis and AMBER assays, pif1-NLSΔ cells were not as defective as pif1Δ for suppression of dna2Δ lethality (Fig 8). Lethality of dna2Δ is thought to result from the persistence of long flaps produced by Pif1 during Okazaki fragment maturation. We propose that even a few long flaps resulting from the nuclear localization of small amounts of Pif1-NLSΔ protein is sufficient to kill cells lacking Dna2. Nevertheless, formation of some viable pif1-NLSΔ dna2Δ spores suggests that at least some of these cells had no nuclear Pif1-NLSΔ protein.

Nuclear entry of Pif1 in the absence of the ⁷⁸¹KKRK⁷⁸⁴ motif

Given that residual levels of both BIR and Okazaki fragment maturation occurred in pif1-NLSΔ cells (Figs 5,6, and 8), nuclear entry of some Pif1 must occur even in the absence of the ⁷⁸¹KKRK⁷⁸⁴ motif. Likewise, ChIP assays detected Pif1-NLSΔ protein bound to two of two known nuclear target sites, although at both sites, Pif1 bindings was ~5-fold lower than in WT cells (Fig 7). In contrast, Pif1-NLSΔ binding to a mitochondrial site was not reduced (Fig 7).

We envision at least three possibilities for how Pif1 enters the nucleus in pif1-NLSΔ cells. The most likely mechanism is that a fraction of the 98kDa Pif1-NLSΔ diffuses into the nucleus, as there is no sharp molecular weight cut off for passive protein import [17]. Another possibility is that small amounts of Pif1 can enter the nucleus via its association with another protein that has its own NLS. Finally, a less active NLS (for example the bipartite NLS identified by cMapper; see Results) may function in a subset of cells. Nonetheless, our data suggest that nuclear entry of Pif1 is heavily reliant on the ⁷⁸¹KKRK⁷⁸⁴ motif. We note that highly sensitive genetic assays are likely better able to detect low levels of nuclear entry compared to cytological or physical assays (such as telomere length). Moreover, small amounts of an enzyme are more likely than small levels of a structural protein to provide detectable function.

New separation of function pif1 alleles with impaired nuclear function

A pif1Δ strain is not convenient for analysis of nuclear Pif1 functions because of its slow growth. In addition, the defective mitochondrial genomes in different pif1Δ strains are not necessarily identical, and impaired mitochondrial function may result in secondary phenotypes that are not due directly to lack of Pif1. Hence, a separation-of-function form of Pif1 that is defective for nuclear, but proficient for mitochondrial functions, would be valuable. As shown here, in two sensitive BIR assays, pif1-NLSΔ cells had even more residual BIR activity than pif1-m2 (Figs 5 and 6). The fact that the doubly mutant pif1-m2+NLSΔ allele was more defective than either single mutant in sensitive BIR assays is consistent with the two mutations acting at different steps in Pif1 biogenesis. That is, the pif1-m2 allele results in reduced translation specifically of the nuclear form of Pif1 [4], while the NLSΔ allele reduces nuclear entry of Pif1. Even though there is residual nuclear protein by the most sensitive assays even in pif1-m2+NLSΔ cells, unlike pif1Δ, all three of the partial loss of function pif1 alleles had WT or near WT growth rates and maintained mitochondrial function. Moreover, even though the pif1-m2+ NLSΔ allele was not a null in the most sensitive BIR assays, it was more defective than either single mutation. Thus, a pif1-m2+ NLSΔ strain is currently the best one for studying nuclear functions of Pif1.

Yeast strains and growth conditions

To assess the effects of mutant PIF1 alleles on dna2Δ lethality and on mitochondrial proficiency, we used two yeast diploid strains YCG57 or YCG59 (both derived from W303 [40]) that had the following genotypes: YCG57 (MATa/MATa leu2-3,112/leu2-3,112, trp1-1/trp1-1, can1-100/can1-100, ura3-1/ura3-1, ade2-1/ade2-1, his3-11/his3-11, PIF1/pif1::NatMX6), YCG59 (MATa/MATa leu2-3,112/leu2-3,112, trp1-1/trp1-1, can1-100/can1-100, ura3-1/ura3-1, ade2-1/ade2-1, his3-11/his3-11, PIF1/pif1::NatMX6, DNA2/dna2::KanMX6) [19] (see S1 Table for all genotypes of yeast strains).

The efficiency of BIR and telomere lengths were determined using the AM1003 [27] and its derivatives. These strains are disomic for chromosome III and express the HO endonuclease under the control of a galactose-inducible promoter. The rate of BIR-associated mutagenesis was assessed using derivatives of AM1003 containing a frameshift reporter gene (lys2::A₄) inserted into the donor chromosome 16kb centromere-distal to MATα-inc (AM1291 and its derivatives) [29]. This mutant allele of LYS2 gene has an insertion of 61bp, leading to a Lys^- phenotype due to +1-bp frameshift. A Lys⁺ phenotype is usually restored by a 1bp deletion in the reporter.

The construction of pif1-m2 and pif1Δ derivatives of AM1291 (AM2061 and AM2191, respectively) was described previously [28].

To insert other pif1 mutant alleles, we first replaced the hygromycin-resistance gene (HPH) located at HMR in AM1003, AM1291, and AM2061 with KANMX (G418 resistance marker) to create AM5640, AM5978, and AM6121 respectively. To introduce pif1-NLSΔ, these strains were co-transformed with an oligo OL5029 (S6 Table) and plasmid pRL10 (see S7 Table; [41]) that contains a region for targeting the NLS region that is complementary to the 50bp 5’ of the Pif1 NLS and the 50bp 3’ of the NLS region but lacks the NLS itself. Deletion of the PIF1 NLS region in AM6007 (AM1003 derivative), AM6032 (AM1291 derivative), and AM6201 (AM2061 derivative) was confirmed by PCR and sequencing. To insert the SV40-NLS into the strains containing pif1-NLSΔ, the SV40-NLS region was PCR amplified from pCG82 (see S7 Table) using the following primers: OL5032 (S6 Table) homologous to PIF1 region located centromere proximal to NLS, and OL5033 (S6 Table) where the small letters correspond to the SV40-NLS sequence of pCG82 and the capital letters represent homology to the region flanking the PIF1 gene (located centromere distal from PIF1). This PCR product was co-transformed with pRL11 (CRISPR-Cas9 plasmid that contains a region corresponding to 854 aa of Pif1 for CRISPR-Cas9 targeting; see S7 Table). The resulting strains containing the SV40-NLS are AM6057 (AM6007 derivative) and AM6060 (AM6032 derivative).

For ChIP analysis, Pif1 alleles were tagged with 13xMyc epitopes that were amplified from the pFA6a-13XMyc-KanMX6 plasmid [33] by using the following primers: RL216 and RL217 (S6 Table). The resulting PCR product was co-transformed with pRL11 into AM6007, AM1003, and AM6201 to generate strains AM6737, AM6740, and AM7068, respectively (S1 Table). In addition, primers RL367 and RL217 were used to amplify SV40 NLS and 13xMyc together, and the PCR product was co-transformed with pRL11 into AM6007 to generate AM7077 (S1 Table).

The constructs were confirmed by Sanger sequencing, and the level of Pif1 in these strains was analyzed by western blot. The construction of diploids used to analyze the suppression of dna2Δ lethality by pif1-NLSΔ involved the following steps: (i) haploid MATα-inc strains were produced by plating AM6007 and AM1003 on YEP-Gal media followed by selection of Ade^-red Leu^- cells to obtain AM6867 (pif1-NLSΔ) and AM6870 (PIF1) (see S1 Table). (ii) KANMX in AM6867 was replaced by HPH to obtain AM6872. (iii) NP265 (MATa pif1Δdna2Δ, see S1 Table) was a gift from Dr. Grzegorz Ira lab. AM6872 and AM6870 were crossed to NP265 to generate diploid strains (AM6902 and AM6900, respectively).

Plasmids

The empty vector used for the analyses of a mitochondrial function and of dna2Δ suppression was pRS414 plasmid (ARS CEN6 TRP1) [42]. pCG17 is a derivative of pRS414 that contains PIF1 under control of its endogenic promoter and that was constructed in the following steps: (i) the PsPX1/AgeI fragment containing PIF1 with a carboxy-terminal 3xFLAG tag under control of RRM3 promoter was cloned into pRS414 digested with PsPX1 and AgeI to produce pMB282 [9]; (ii) the RRM3 promoter in pMB282 was replaced with endogenous promoter of PIF1 (using digestion with PsPX1 and AgeI) to create pCG17 [19]. pCG80 (see S7 Table), which carries the pif1-NLSΔ allele, was generated from pCG17 by deleting the sequence corresponding to amino acids ⁷⁸¹KKRK⁷⁸⁴ of Pif1. pCG82 was constructed from pCG80 by inserting the region of SV40 that encodes the SV40 NLS for T-antigen (amino acids ¹²⁶PKKKRKV¹³²) into the C-terminus of pif1-NLSΔ. Plasmids were introduced into yeast cells via lithium acetate transformation [43]. CRISPR-Cas9 plasmids (pRL10 and pRL11), that contain a target region for NLS of Pif1 and carboxy-terminal of Pif1, respectively, were constructed as described [41] and used in construction of pif1-NLSΔ (AM6007 and AM6032), pif1-m2+NLSΔ (AM6201), and pif1-NLSΔ+SV40 (AM6057 and AM6060) strains, respectively. pFA6a-13xMyc-KanMX6 plasmid [33] was used as a template to copy 13xMyc to tag various pif1 alleles for ChIP analysis.

Media and growth conditions

Depending on the experiment, cells were grown in rich medium YEPD (1% yeast extract, 2% peptone, and 2% glucose) or synthetic drop-out medium lacking tryptophan (Sc-Trp [44]) with glucose (2%) or raffinose (3%). For experiments assessing BIR efficiency or BIR-associated mutagenesis, strains were grown in synthetic drop-out medium lacking leucine with glucose (2%) (Sc-Leu [44]) in rich medium containing lactic acid (3%) instead of glucose (YEP-Lac [45]), followed by addition of galactose directly to the media (to a final concentration of 2%), or plated and on rich medium where glucose was replaced by galactose (2%) (YEP-Gal) [45]. The BIR outcomes were characterized by replica plating on synthetic drop-out media lacking leucine or adenine with glucose (2%) (Sc-Leu and Sc-Ade respectively) [45]. The BIR-associated mutagenesis was assessed by plating on synthetic drop-out medium lacking both adenine and lysine (Sc-Ade, Lys) [45]. To induce meiosis, diploid cells were sporulated by using pre-sporulation medium (YPA, similar to YEPD but potassium acetate was added to 2% instead of glucose) followed replica plating to sporulation medium (SM) that contained 2% potassium acetate [46].

Identification of the candidate Pif1 NLS motif and construction of Pif1 NLS alleles

The cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/) was used to predict putative nuclear localization signals (NLS) within Pif1 [23]. The core of the functional NLS was narrowed down to a stretch of four basic amino acid residues (⁷⁸¹KKRK⁷⁸⁴). Deletion of these four amino acid motifs was generated in pCG17 using Quik Change Lightning Site-directed Mutagenesis (Agilent Technologies) to create pCG80. A heterologous NLS (¹²⁶PKKKRKV¹³²) from the SV40 T antigen [25] was introduced into the carboxy-terminal end of pCG17 and pCG80 using gBlocks gene fragments (Integrated DNA Technologies, IDT) and the Gibson Assembly® method (New England Biolabs, NEB). pif1-NLSΔ alleles were verified by both restriction enzyme digestion using AvaI and NotI (NEB) and DNA sequencing (GeneWiz).

Immunoblot analysis

Yeast total protein extracts were prepared using trichloroacetic acid (TCA) precipitation [47]. Briefly, 10 ml of mid-log phase cells were collected and resuspended in 20% TCA. The cells were lysed with glass beads and vortexing (three 1 min cycles, 4°C) using the FastPrep-24homogenizer (MP Biomedicals). Two different variations of the method were used after this point:

Variation A (used for western blot analysis shown in Fig 1C). The glass beads were washed with 5% TCA and the precipitated proteins were collected by centrifugation. The protein was resuspended in 1X Laemmli buffer (150 mM Tris pH 6.8, 6% SDS, 30% glycerol, 0.3% bromophenol blue, 15% β-mercaptoethanol) and 1M Tris base, then boiled for 5 min. The proteins were resolved on a 7% SDS-PAGE and transferred to nitrocellulose membrane (GE Healthcare). FLAG-tagged Pif1 proteins were detected using mouse anti-FLAG M2 antibody (Sigma-Aldrich) and visualized with HRP-conjugated anti-mouse secondary antibody (Bio-Rad) using standard ECL detection reagents (GE Healthcare) and imaged using the AlphaImager HP system (Protein Simple).

Variation B (used for western blot analysis shown in Fig 7A). The precipitated proteins were collected by centrifugation and washed by 0.5M Tris-HCl (pH 8.0). The protein was resuspended in 2x loading buffer (10mM Tris pH 6.8, 4% SDS, 20% glycerol, 0.2% bromphenol blue, 10% β-mercaptoethanol) and 0.5M Tris base (pH 8.0), then boiled for 5 min. The proteins were resolved on 4–20% SDS-PAGE and transferred to nitrocellulose membrane (Amersham #10600002). MYC-tagged Pif1 proteins were detected using antibody (Sigma M4439) and visualized using IRDye 800CW Goat anti-Mouse (#926–32210) from LI-COR. Blots were imaged using an LI-COR Odyssey-Fc Imager.

Analyzing mitochondrial proficiency

100 ng of plasmid DNA was transformed into a heterozygous PIF1/pif1Δ::NatMX6 diploid strain YCG57 (see S1 Table). After sporulation and dissection, haploid pif1Δ spores carrying TRP1 plasmids were selected and grown to saturation in 5 ml Sc-Trp medium. As previously described [19], serial dilutions were spotted onto Sc-Trp medium with 2% glucose or 3% glycerol and incubated at 30°C for at least 3 days.

Telomere length analysis

Genomic DNA was purified from yeast strains as described in [45], and 1.0–1.5ug of this DNA was digested with XhoI restriction enzyme (NEB). Following XhoI digestion, DNA was separated by gel electrophoresis in 1.2% agarose gel in TBE buffer, transferred to a nylon membrane (GE Healthcare), and hybridized (as described [45]) to a ³²P-labelled probe specific to Y’ pre-telomeric region [48] that was prepared as described in [45]. Blots were imaged using an Azure Sapphire Biomolecular Imager. All strains containing various alleles of PIF1 were propagated on YEPD plates for at least 100 generations to allow time for establishment of telomere lengths characteristic for each particular PIF1 allele.

BIR efficiency assay

Cells were grown in 5ml of liquid Sc-Leu media for approximately 24 hours at 30°C until saturation. Cells were then transferred to YEP-Lac media at ~1-3x10⁶ cells/ml and grown for ~ 16 hours until cell density was about ~1-2x10⁷ cells/mL. Then cells were plated on YEP-Gal and YEPD plates (at ~50 cells per plate). Plates were incubated for 5–7 days, and then replica plated onto Sc-Ade or Sc-Leu. The frequencies of BIR, gene conversion (GC), half-crossover (HC), and chromosome loss (CL) events were calculated based on respective fractions of Ade⁺ Leu^–, Ade⁺ Leu⁺, Ade^-white Leu^-, Ade^-red Leu^- outcomes among all DSB repair events (as described in [45]). Since Ade⁺ Leu⁻can be generated by both BIR and GCRs, the fraction of GCRs among Ade⁺ Leu⁻was estimated by analyzing the representative number of these repair outcomes using CHEF gel electrophoresis (similar to [45]). Due to random segregation of chromosomes, half of the HC products segregate with an intact copy of full-length chromosome III leading to formation of repair products (HC-II) that are genetically indistinguishable from BIR [27]. Therefore, it was assumed that the number of HC-II events is equal to the number of HC-I (Ade^-white Leu^- events). Consequently, the number of BIR was adjusted by subtracting the number of HC-II.

BIR mutagenesis assay

Cells were grown at 30°C in 5ml of Sc-Leu for ~24 hours, diluted 20-fold with YEP-Lac, and then grown for ~16 hours until cell density reached ~1-2x10⁷cells/mL. BIR was induced by addition of 20% galactose to a final concentration of 2%, and cells were incubated for 7 hours after galactose addition. The rate of mutagenesis that occurs during BIR-associated DNA replication was determined by plating cells on Sc-Ade, Lys and Sc-Ade (see [45] for the details of rate calculation). The resulting mutagenesis rate was compared to the rate of mutagenesis that occurs during a normal S phase, calculated by plating cells to the same media prior to galactose addition (see [45] for the details of calculation).

AMBER (assay for monitoring BIR elongation rate) to detect BIR-associated DNA synthesis

Cells were grown overnight at 30°C in 5ml of Sc-Leu, transferred (1:100 dilution) to YEP-Raffinose and incubated for 16hrs until cell density of ~5x10⁶ cells/ml. Then, 50ml of cells were collected (0hr) and stored at -80°C. Galactose was added to the culture to a final concentration of 2% to induce HO endonuclease. During the following 10hrs of incubation at 30°C, ~30ml of cells were collected every hour and stored at -80°C. Genomic DNA was purified and quantified as described for AMBER assay in [31]. For droplet digital PCR (ddPCR) the reaction mix was assembled as follows: 6ul H₂O, 1ul of each primer set (one set of primers for ACT1, and another–for target position), 10ul of ddPCR supermix (Biorad, #1863026) and 2ul DNA (0.1ng/uL) were added and mixed thoroughly by vortexting. ddPCR was conducted and analyzed as described in [31]. The results were presented as Boltzmann sigmoidal curve. Mean values of target to reference (ACT1) loci ratios were calculated by Poisson distribution based on 20,000 droplets with error bars representing upper and lower Poisson 95% CI.

Assaying the effects of PIF1 mutants on the viability of dna2Δ cells

A qualitative assay for characterization of dna2Δ lethality suppression by different alleles of pif1 has been previously described [19]. In particular 500 ng of plasmid DNA (plasmids included different alleles of PIF1) was transformed into a heterozygous PIF1/pif1Δ::NatMX6 DNA2/dna2Δ::KANMX6 diploid YCG59 (see S1 Table). After sporulation and dissection, haploid pif1Δdna2Δ spores carrying TRP1 plasmids were selected and grown in 5ml Sc-TRP medium. The cells were plated onto non-selective rich medium YEPD and onto Sc-TRP and grown at 30°C for 3–4 days. Growth on selective Sc-TRP medium indicates lack of Pif1 nuclear function, as PIF1 deficiency suppresses the lethality of dna2Δ cells [6]. For quantitative assay of lethality suppression, diploid cells that were DNA2/dna2::URA3 and also either PIF1/pif1::KANMX or Pif1-NLSΔ/pif1::KANMX were sporulated and the resulting spores were analyzed by either tetrad analysis (similar to [46]) or by random spore analysis (RSA). For RSA, diploid cells were grown in 5ml of liquid YEPD at 30°C overnight and transferred to 50ml of liquid YPA media (1:10 dilution ratio) and incubated for ~24hr at 30°C. Cells were collected and transferred into 50ml of liquid SM media and incubated for at least 72hrs at 30°C. Cells were spun down and resuspended in 5ml of sterile water. An equal volume of diethyl ether was added to samples, and the mixture was thoroughly mixed for at least 10 minutes at room temperature. Cells were centrifuged and washed by 30ml of sterile water four times, serially diluted, plated on YEPD plates, and incubated for 7days at 30°C. The phenotypes of meiotic outcomes were classified by replica plating onto Sc-Ade, Sc-Ura, and YEPD+G418 (0.5g/L).

Chromatin Immunoprecipitation (ChIP) analysis

To perform ChIP assay, 13 repeats of the Myc epitope were inserted at the C-terminus of both PIF1 and pif1-NLSΔ. The levels of Pif1 protein expressed in these two strains was determined by western blot analysis. ChIP analyses was performed as described previously [12] with several modifications. Specifically, yeast cells were grown to a density between 5.0x10⁶ and 1x10⁷ cells/ml in YEP-raffinose and HO-generated DSBs were induced by addition of 20% galactose (to a final concentration of 2%). 40ml of cells were collected before (0h) and after (4h) DSB induction and proteins were crosslinked by the addition of formaldehyde (Sigma #252549) to a final concentration of 1%. Cells were incubated at room temperature for 10 min followed by addition of glycine (125mM final concentration). Cells were incubated for 5 min, and samples were then stored at -80°C. Cells were lysed with glass beads, and DNA was sheard by sonication to an average size of 0.5kb. Extracts were divided into IP and input samples (9:1 ratio). IP samples were incubated with anti-Myc antibody (Sigma M4439) overnight at 4°C. Proteins were bound to Dynabeads Protein G (Invitrogen 1004D) and taken through a series of washes and then reverse crosslinked and eluted. Samples were treated with proteinase K followed by phenol DNA extraction and DNA precipitation. Protein enrichment was measured by quantitative-PCR (qPCR) analysis from the input (1:10 dilutions) and IP (undiluted) samples. The following primers were used for qPCR analysis (see S6 Table for details): RL220, RL221 (for MAT locus), RL251, RL252 (for CEN13), RL304, RL305 (for mtDNA). Both input and IP signals were normalized by using the following primers: RL218, RL219 (ACT1 locus) used for normalizing data for MAT, and RL196, RL197 (YBL028C locus) used for normalizing data for CEN13 and mtDNA.