Diagram showing the diverse in vivo functions of the ScPif1 DNA helicase. Figure is inspired by an earlier depiction of ScPif1 functions (). See introduction for references for these functions.

(A) Schematic of domains of the ScPif1 DNA helicase. The helicase core domain is in white. The red bars within the core domain are the conserved helicase motifs, and the star indicates the position of the Pif1-family Signature Motif (SM), which is located between helicase motifs II and III. The purple and blue domains are the amino and carboxyl regions of ScPif1, respectively, which vary in size and sequence among different Pif1 family helicases. As indicated, the amino terminus of ScPif1 is 237 amino acids, the helix domain is 508 amino acids and the carboxyl region is 114 amino acids. (B) Sequence alignment of Pif1-family SM from different organisms (Al, Aspergillus lentulus; Bs; Bacteroides spp; Ca, Candida albicans; Cg, Candida glabrata; Cl, Canus lupus familiaris; Hs, Homo sapiens; Mm, Mus musculus; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Xl, Xenopus laevis; Xt, Xenopus tropicalis). Protein sequences were obtained from NCBI (htts://www.ncbi.nlm.nih.gov), aligned using Clustal Omega and analyzed using Unipro UGENE. The consensus row at the top indicates the most conserved residue at each position or shows a ‘+’ when two or more residues are equally abundant. Residues are colored in accordance to their physiochemical properties: aliphatic/hydrophobic (pink), aromatic (orange), positive charge (blue), negative charge (red), hydrophilic (green), proline/glycine (magenta) and cysteine (yellow). (C) SM mutations in ScPif1 generated and analyzed in this study. Mutations include alanine substitutions of conserved residues (I), large and small deletions that are represented by dashed lines (II), and successive amino acid substitutions (III). (D) Structure of a truncated ScPif1 helicase (PDB ID: 5O6D, amino acids 237–780) () rendered and modified using Pymol (). Secondary structures are colored as follows: helix (teal), loop (beige) and sheet (pink). The ScPif1 SM, which is shown in yellow, forms an α-helix with an extended loop.

Analyzing ScPif1 SM mutants for mitochondrial proficiency. As indicated in the cartoon on the left of the figure, cultures were 10-fold serially diluted left-to-right and spotted on selective media containing glucose or glycerol. Respiratory deficient cells grow on media containing glucose but not on media containing glycerol.

(A) Analysis of telomere lengths in cells expressing different PIF1 alleles. Genomic DNA was isolated from three or more independent isolates from each of the 14 SM mutant strains as well as from WT, pif1Δ and catalytically dead pif1-K264A cells (in this figure two representative examples are shown for 9 of the 17 strains examined). DNA was digested with XhoI, separated on 1% agarose gels and analyzed by Southern hybridization using a radiolabeled sub-telomeric Y’ probe (). The lanes contain DNA from WT (lanes 1 and 2), SMΔ6 (lanes 3 and 4), SM15A (lanes 5 and 6), I371A (lanes 7 and 8), G369A (lanes 9 and 10), G370A (lanes 11 and 12), Q372A (lanes 13 and 14), BsPif1-SM (lanes 15–16) and pif1Δ (lanes 17 and 18). (B) Quantitation of telomere lengths. To obtain the number of base pairs of TG_1–3 telomeric DNA, 875 bp, the amount of Y’ DNA in the terminal XhoI restriction fragment () was subtracted from the size of the terminal fragments. The average telomere length was determined for at least three biological isolates per strain. The white lines represent the peak DNA signal that indicates the average length of the Y’ telomeres. Telomeres were classified in Table as having WT (320 ± 12 bp), intermediate (357 ± 11 bp) or null (441 ± 53 bp) telomere lengths. Error bars are standard deviations. Given that loss of ScPif1 function results in longer telomeres, a one-tailed student t-test was used to compare the mean telomere lengths between WT and ScPif1 mutants to determine if the increase in telomere lengths observed in the SM mutants was statistically significant; NS, not significant, *P<0.05, **P<0.005.

Analysis of the ability of ScPif1 SM mutants to suppress the lethality of a dna2Δ cells. Plasmid-borne WT and SM alleles were introduced into pif1Δ dna2Δ cells and analyzed for growth on selective media as indicated in the cartoon. WT alleles are unable to suppress the lethality of dna2Δ cells. Images shown are cells plated on selective media lacking tryptophan; growth indicates null ScPif1 function.

(A) Schematic of gross chromosomal rearrangement (GCR) assay (). (B) Bar graph shows GCR rates of WT ScPif1, P367A, SMΔ6, G369A, G370A, Q372A and SM6A mutants. The average rate in cells expressing WT ScPif1 was 1.20 × 10⁻⁷ events per cell division and the average rates in mutant cells expressing single alanine substitutions were similarly low. The GCR rate for cells expressing P367A, an allele that was WT in all in vivo assays, was 6.68 × 10⁻⁸. The GCR rate for cells expressing G369A, G370A and Q372A, alleles that exhibited an intermediate telomere phenotype, were 5.85 × 10⁻⁸, 5.79 × 10⁻⁸ and 5.36 × 10⁻⁸, respectively. In contrast, the average rates in mutant strains SMΔ6 and SM6A were almost 50-fold higher compared to WT. Experiments were done on at least three biological replicates. Error bars show standard deviations.

The SM of ScPif1 is required for DNA dependent ATPase activity in vitro. (A) SDS-PAGE analysis of the purified ScPif1 WT and mutant ScPif1 proteins prepared in Escherichia coli. The asterisk indicates a contaminating band that co-purifies with full-length Pif1 variants with mutations within the SM. (B) DNA-dependent ATPase activity of Pif1 (N- and C-terminal His₆-tagged) and the single-point mutant variants G370A and I371A. (C) Time course of ATP hydrolysis, monitoring the absorbance of NADH, of Pif1 and its SMaa6Δ and SM6A variants. (D) Time courses of ATP hydrolysis of Pif1^238-859 and its SM6G variant. (E) Forked-DNA binding activity of Pif1^238-859 and its SM6G variant.