The role of Pif1p, a DNA helicase in Saccharomyces cerevisiae, in maintaining mitochondrial DNA

Mitochondrion 7 (2007) 211–222 www.elsevier.com/locate/mito

The role of Pif1p, a DNA helicase in Saccharomyces cerevisiae, in maintaining mitochondrial DNA Xin Cheng a, Stephen Dunaway b, Andreas S. Ivessa a

a,*

Department of Cell Biology and Molecular Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange Avenue, Newark, NJ 07101-1709, USA b Biology Department, Drew University, 36 Madison Avenue, Madison, NJ 07940, USA Received 22 August 2006; received in revised form 22 November 2006; accepted 27 November 2006 Available online 9 December 2006

Abstract Mitochondrial DNA (mtDNA) is highly susceptible to oxidative and chemically induced damage, and these insults lead to a number of diseases. In Saccharomyces cerevisiae, the DNA helicase Pif1p is localized to the nucleus and mitochondria. We show that pif1 mutant cells are sensitive to ethidium bromide-induced damage and this mtDNA is prone to fragmentation. We also show that Pif1p associates with mtDNA. In pif1 mutant cells, mtDNA breaks at specific sites that exhibit Pif1-dependent recombination. We conclude that Pif1p participates in the protection from double-stranded (ds) DNA breaks or alternatively in the repair process of dsDNA breaks in mtDNA.  2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: Mitochondrial DNA; DNA helicase; DNA replication; DNA repair; DNA damage

1. Introduction Mitochondrial DNA (mtDNA) encodes several components of the electron transport chain along with several transfer and ribosomal RNAs necessary for the synthesis of mitochondrial proteins (Foury et al., 1998). The genes of the mitochondrial genome are essential for the execution of cellular respiration and oxidative phosphorylation. Many mammalian cell types, like neurons or heart muscle cells, cannot afford to lose mitochondrial function. Dysfunctional mitochondria cause these cells to undergo apoptosis because glycolysis alone fails to cover all the cellular energy demands (Van Houten et al., 2005). Non-functional mitochondria may contribute to the development of several mitochondrial diseases which often result from mutations in mtDNA (Taylor and Turnbull, 2005). These diseases include certain cancers, heart diseases, neurodegenerative diseases, eye diseases, and Type II diabetes.

*

Corresponding author. Tel.: +1 973 972 2015; fax: +1 973 972 7489. E-mail address: [email protected] (A.S. Ivessa).

mtDNA is not protected by histones and is therefore up to 10-times more susceptible to damage by environmental carcinogens, therapeutic drugs, and endogenous reactive oxygen species (ROS) than nuclear DNA (Bandy and Davison, 1990; Xia et al., 2004). These features might explain why mutations in mtDNA accumulate and why mitochondrial function declines during aging (Singh, 2004; Trifunovic et al., 2004). Major damage in mtDNA results from the influence of endogenous ROS that can be corrected by the base excision repair pathway, BER. The well-studied mitochondrial BER pathway is similar to the nuclear BER system (Mandavilli et al., 2002; O’Rourke et al., 2002; Stevnsner et al., 2002; Larsen et al., 2005). Proteomic analysis of yeast mitochondria, however, has identified members of additional repair pathways such as mismatch repair (Mlh1p), nucleotide excision repair (Rad23p), translesion repair (Rad18p), and recombinational repair (Mre11p, Rad50p) (Sickmann et al., 2003; Larsen et al., 2005). However, it is not yet understood whether these pathways are functionally at work in the mitochondria, as they are in the nucleus. One protein thought to be involved in the repair of damaged mtDNA is the DNA helicase Pif1 (Foury and Lahaye,

1567-7249/$ - see front matter  2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2006.11.023

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1987; Schulz and Zakian, 1994; O’Rourke et al., 2002). In yeast, PIF1 is encoded by a single, non-essential nuclear gene. Pif1 protein (Pif1p) has nuclear and mitochondrial targeting signals and is localized to both the nucleus and mitochondria (Lahaye et al., 1991; Zhou et al., 2000). The nuclear form of Pif1p acts at multiple chromosomal loci and has multiple functions. Pif1p catalytically inhibits telomerase activity, replication fork progression in ribosomal DNA, and prevents gross-chromosomal rearrangements throughout the yeast nuclear genome (Schulz and Zakian, 1994; Ivessa et al., 2000; Myung et al., 2001). Although Pif1p has 5 0 –3 0 DNA helicase activity (Lahaye et al., 1991), recent findings show that Pif1p also unwinds DNA/RNA hybrids with the same polarity (Boule et al., 2005). Since Pif1p inhibits telomerase activity, Pif1p may regulate telomerase activity by a temporal displacement of the RNA component from telomeric DNA. The nuclear form of PIF1 interacts genetically with the DNA helicases DNA2 and SGS1, the DNA polymerase POL32, and with the topoisomerase TOP3 (Budd et al., 2006; Wagner et al., 2006). These genetic interactions suggest that Pif1p has roles in the formation/maturation of Okazaki fragments and/or in repair of damaged DNA (e.g., double-stranded DNA (dsDNA) breaks). Pif1p was first described as a mitochondrial DNA helicase (Lahaye et al., 1991). Although mutant strains that lack Pif1p can still be grown on medium containing nonfermentable carbon sources such as glycerol, these strains exhibit increased mtDNA instabilities exhibited as point mutations, deletions (q) or loss (q0) of mtDNA (Van Dyck et al., 1992; O’Rourke et al., 2002). This phenotype results in the appearance of small colonies (i.e., petite colonies) on non-fermentable carbon sources. Pif1p stimulates the recombination frequency between full-length, functional mtDNA molecules and various truncated versions of mtDNA that have tandemly arrayed repeat units. Although these analyses suggest that Pif1p only affects recombination in certain areas in mtDNA, it was concluded from these studies that Pif1p may recognize topological structures in mtDNA rather than specific sequences (Foury and Lahaye, 1987; Lahaye et al., 1991). Pif1p is highly related to its yeast homolog, the DNA helicase Rrm3p and to homologues in other species such as Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens (Bessler et al., 2001). Pif1p and Rrm3p form their own subgroup among the 137 helicases and helicaserelated proteins in bakers yeast (Shiratori et al., 1999). Here, we show that Pif1p is required to maintain mtDNA in the presence of the genotoxic chemical ethidium bromide (Et–Br). When we examined mtDNA replication intermediates in WT and Pif1p-lacking pif1D cells, we observed that mtDNA breaks at specific sites under natural conditions. Since Pif1p associates with the entire mitochondrial genome, its effects on mtDNA are likely direct. We propose that Pif1p either prevents or repairs dsDNA breaks in mtDNA.

2. Materials and methods 2.1. Media, strains, and plasmids The strains were grown in 3% glycerol-containing medium to select for the cells with functional mitochondria. In general, media were prepared as described in Rose et al. (1990). All strains are in the yPH499 strain background except for the strain that is free of mtDNA (used in Fig. 5) (Sikorski and Hieter, 1989). The yeast strain lacking mtDNA (generously provided by Dr. C. Suzuki, UMDNJ) has the following background: MATa leu2 ura3 rme1 his4. The same strategy as described in Schulz and Zakian was applied to introduce the m1 and m2 mutations, respectively, into the PIF1 gene (Schulz and Zakian, 1994). The PIF1 gene was tagged at its C-terminus with a 13myc tag (Longtine et al., 1998). The strain is able to complement the Et– Br sensitivity (such as shown in Fig. 1) and the resulting protein migrates at the expected size of 114 kDa as assayed by Western blotting technique. Yeast strains were grown at 30 C. For the Et–Br sensitivity test, Et–Br was added to the medium to a final concentration of 1 lg/ml culture medium. Transformation into yeast was done by standard procedures (Schiestl and Gietz, 1989). 2.2. Test-spot assay and serial dilution series to examine Et– Br sensitivity For the test-spot assay, yeast cells were grown to saturation in YEP medium in the presence of 3% glycerol, 10-fold serial dilutions were spotted on YEP agar plates containing either 3% glycerol or 2% glucose. Et–Br was added to the final concentration of 1 lg/ml culture medium. 2.3. Examination of mtDNA Cells were grown for 24 h in YEP medium containing 3% glycerol to examine mtDNA under the influence of Et–Br. Et–Br was added where indicated to a final concentration of 1 lg/ml culture medium. Total genomic DNA was purified using a commercial-available purification kit (Epicentre, WI), separated on a 0.8% agarose gel, and analyzed by Southern technique (Southern, 1975). The membrane was probed with DNA fragments encoding the mitochondrial gene cox1 to detect mtDNA (generously provided by Dr. R. Butow, U of Texas Southwestern). The mitochondrial cox1 probe was removed, and the membrane was hybridized to DNA fragments encoding the chromosomal probe CEN4. 2.4. Replication 2D gel technique To examine intermediates in replication and recombination, the two-dimensional (2D) neutral–neutral gel electrophoresis technique was applied essentially as previously described (Ivessa et al., 2000; Ivessa et al., 2002; Ivessa et al., 2003). Yeast mtDNA for 2D gel analysis was

X. Cheng et al. / Mitochondrion 7 (2007) 211–222

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Fig. 1. Absence of the mitochondrial form of Pif1p causes sensitivity to the genotoxic drug ethidium bromide (Et–Br). Haploid strains were grown in glycerol-containing medium. Equal numbers of cells were spotted in ten-fold serial dilutions on plates containing either glucose (A and C), or glycerol (B and D), and Et–Br (1 lg/ml) was included as indicated (C and D). Wild-type and mutant Pif1 proteins are schematically shown in (E).

prepared as previously described (MacAlpine et al., 1998; Noguchi et al., 2003). Briefly, yeast cells of an early logarithmic growth phase were treated with zymolyase in an osmotic stabilizing buffer (glycerol) to degrade the cell wall. Cells were lysed by the addition of the detergent N-laurylsarcosine and proteinase K. MtDNA was separated from chromosomal DNA by CsCl gradient centrifugation as previously described (Huberman et al., 1987). MtDNA was digested either with the restriction enzymes BamHI or EcoRV. DNA fragments of the var1 and cox1 genes (generously provided by Dr. R. Butow, U of Texas Southwestern), respectively, were used as detection probes in the Southern analysis. The amount of replication intermediates and un-replicated DNA was determined by phosphor-imager analysis using the software imagequant. 2.5. Mitochondrial DNA immunoprecipitation technique The chromatin immunoprecipitation technique (here: mtDNA immunoprecipitation or mIP) was applied to detect protein–mtDNA interactions as previously described (Hecht et al., 1999; Taggart et al., 2002; Fisher et al., 2004). The procedure optimized for chromosomal DNA was adjusted according to an available protocol for mIP (Hall et al., 2004). In brief, yeast cells were treated with formaldehyde (1% final concentration) for 10 min at RT. Formaldehyde creates crosslinks in vivo between proteins and DNA, but also between proteins itself. The cells were lysed using glass-beads, DNA was sheared into small pieces (range of 0.4–0.7 kb) by ultra-sonication. Commercially available anti-myc antibodies (Clontech, CA) and magnetic IgG beads coated with protein G (Invitrogen, CA) were used to immunoprecipitate myc-tagged Pif1 protein. Magnetic beads of this type has been shown to lower the non-specific background significantly compared to agarose beads (Taggart et al., 2002; Fisher et al., 2004). The formaldehyde crosslink was reversed, and the DNA was purified using a commercially available PCR clean up kit (Qiagen, CA). Specific DNA

fragments were amplified by PCR. Thermocycling conditions were as follows: 2 min at 95 C; 30 cycles of 30 s at 94 C, 30 s at 52 C, and 80 s at 72 C; followed by a 4 min extension at 72 C. The following primers were used (in parenthesis: expected size of PCR product): For ARO1 (370 bp): F ARO1 5 0 -TGA CTG GTA CTA CCG TAA CGG TTC, R ARO1 5 0 -GAA TAC CAT CTG GTA ATT CTG TAG TTT TGA C; cox1 (698 bp): F mtDNA 14718 5 0 -GGA ACA GGT AAA TTC, R mtDNA 15415 5 0 -TGA ACC TAA TAC CCC A; cob (187 bp): F mtDNA 36604 5 0 -AAC CAT CAT CAA TTA ATT ATT GAT GAA ATA TGG G, R mtDNA 36755 5 0 -TTT GCA TGT AAA TAT CTT AAA ATA TAA CCA TTA TGC; ori6 (249 bp): F mtDNA 44501 5 0 -CCT CAC TCC TCC GGC GTC CTA CTC, R mtDNA 44711 5 0 -ATA ATA ATA AGA AGT TCT AAT TAA TTG TCT CTA TGT CAG G; ori3 (268 bp): F mtDNA 54558 5 0 -TTT TAT TAT GAG GGG GGG, R mtDNA 54810 5 0 -GGG TGG GTG ATT AGA AAC; 21S rRNA (319 bp): F mtDNA 58112 5 0 -GAA TTA GGT TAC TAA TAA ATT AAT AAC AAT TAA TTT TAA AAC C, R mtDNA 58349 5 0 -ATA AAA TAA TCA TTT TCA TAC TTT CCC TTA CGG; cox3 (625 bp): F mtDNA 79260 5 0 -GGT TAT GCC TTC ACC ATG ACC, R mtDNA 79865 5 0 -CAC CTA ACA TAG CTG CTA AC; ori5 (277 bp): F mtDNA 82327 5 0 GTA ATA GGG GGA GGG GGT, R mtDNA 82587 5 0 TTC TAT TGT GGG GGT CCC. The results of the mIP experiments were quantified using the program ImageJ. 3. Results 3.1. Absence of the mitochondrial form of the Pif1p DNA helicase results in sensitivity to the genotoxic drug ethidiumbromide Previous findings suggest that Pif1p either prevents or repairs damage in mtDNA, since pif1D mutant cells are sensitive to UV and exhibit higher mutation frequencies

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in mtDNA (Foury and Kolodynski, 1983; O’Rourke et al., 2002). Here, we investigated whether cells lacking Pif1p are sensitive to the genotoxic drug ethidium-bromide (Et–Br). Et–Br at low concentrations (in the range of 0.1 to 1 lg/ ml) causes fragmentation of mtDNA, formation of a smaller sized mitochondrial genome (q), and at higher concentrations (such as 20 lg/ml) total depletion of mtDNA occurs (q0 mitochondrial genome) (Goldring et al., 1970; Perlman and Mahler, 1971; Bastos Rde and Mahler, 1974; Fukunaga et al., 1984). In general, Et–Br induces damage preferentially in mtDNA and, as it was shown previously, is a useful probe for detecting differences in the organization of mtDNA-protein interactions (Lawrence and Daune, 1976; Chen et al., 2005). We observed that pif1D mutant strains are highly sensitive to Et–Br (final concentration of Et–Br: 1 lg/ml culture medium) only when cells are grown in the presence of nonfermentable carbon sources such as glycerol that require mitochondrial function (Fig. 1D): compare the first row (WT) with the last row pif1D. There is no growth inhibition when the pif1D mutant cells metabolize fermentable carbon sources such as glucose in the presence of Et–Br (Fig. 1C) suggesting that Et–Br does not affect general growth of the pif1D mutant. Since Pif1p is present in both mitochondria and the nucleus, we addressed whether lack of nuclear or mitochondrial Pif1p is responsible for the Et–Br sensitivity. Previously two mutant alleles of PIF1 were described that separate mitochondrial and nuclear functions (Schulz and Zakian, 1994). The pif1-m1 mutation is defined as a substitution of the first available methionine in PIF1 (that functions as the start codon) to alanine (Fig. 1E, middle panel). The resulting Pif1 protein (Pif1-m1p) is predicted to lack the N-terminal mitochondrial targeting signal and is therefore not imported into the mitochondria. The absence of Pif1p in mitochondria causes the appearance of respiratory deficient cells. However, the Pif1-m1 protein is predicted to be imported into the nucleus and fulfills its nuclear functions, since telomeres are maintained at WT length as previously shown (Schulz and Zakian, 1994). The pif1-m2 mutation changes the second available methionine to alanine at amino acid position 40 (Fig. 1E, bottom panel). Since the Pif1-m2 protein is predicted to be present in mitochondria, the pif1-m2 mutant cells show normal respiration activity (Schulz and Zakian, 1994). However, since the nuclear function of PIF1 is lacking in the pif1-m2 mutant strain, telomeres become longer and replication of ribosomal DNA occurs improperly (Schulz and Zakian, 1994; Ivessa et al., 2000). Loss of the nuclear function (pif1-m2) but not of the mitochondrial function (pif1-m1) of PIF1 also suppresses the lethality of a mutant strain lacking the DNA helicase DNA2 (Budd et al., 2006). When we tested the pif1-m1 and pif1-m2 mutant strains for sensitivity to Et–Br, we observed that only the pif1-m1 mutant strain is sensitive to Et–Br on glycerol-containing medium (Fig. 1D, row 2). In contrast, the mutant strain lacking the nuclear function of PIF1 (pif1-m2) grows indis-

tinguishable from WT cells (Fig. 1D, row 3). Taken together, these results suggest that the mitochondrial function of Pif1p is critical to maintain mitochondrial activity in the presence of genotoxic drugs such as Et–Br. Further, we determined the kinetics of the loss of mitochondrial function under the influence of Et–Br. WT and pif1D mutant cells were grown in the presence of glycerol and transferred to a medium that either contained or lacked Et–Br (Fig. 2A). Samples were taken at the indicated times (0, 24, 48, 60, and 72 h). Aliquots of cells were then plated on medium containing glycerol or glucose to assay for the growth of respiratory competent and total cells, respectively. After 24 h of growth in Et–Br-containing medium only about 14% of the pif1D mutant cells were respiratory competent, and after 48 h the amount of respiratory competent cells was reduced to 5% (Fig. 2B, pif1D (+Et–Br)). In contrast, WT cells lose mitochondrial function rather slowly under similar conditions. After 72 h almost 75% of the cells were still respiratory competent (Fig. 2B, WT (+Et–Br)).

Assay for Respiratory Competent Yeast Cells

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Fig. 2. Absence of Pif1p causes functional loss of mitochondria in the presence of ethidium bromide (Et–Br). (A) [1] Meiosis was induced in diploid strains heterozygous for the pif1 deletion pif1D. [2] The haploid spores (Wild-type, WT, and pif1D were placed on plates containing glycerol. [3] Cells derived from individual spores were transferred to liquid medium containing glycerol. [4] Cells were grown to an early log-phase and then aliquots of cells were transferred to Et–Br free glycerol medium or glycerol medium containing Et–Br at a concentration of 1 lg/ml culture medium. [5] Cells (800 cells/plate) were plated at each indicated time point on plates containing glycerol or glucose. (B) A ratio, derived from cells grown on plates containing glycerol (respiratory competent) and on plates containing glucose (total number of cells) was calculated. This ratio reflects the functionality of mitochondria in WT and the various mutant cells. Each value is an average of three experiments (two colonies tested in each experiment); error bars are indicated. In the absence of Pif1p and presence of Et–Br, the function of mitochondria is completely lost after 72 h.

X. Cheng et al. / Mitochondrion 7 (2007) 211–222 Et-Br added to the medium

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1 x 2 x 4x 8 x 1 x 2x 4 x 8x 1 x 2x 4x 8x 1x 2 x 4 x 8x

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Southern blot Probe: Mitochondrial COX1

Genomic DNA Intact mtDNA

4 Degraded mtDNA 2

1

Southern blot Probe: Chromosomal CEN4

Intact chromosomal DNA

Loading control: Genomic DNA (Et-Br stained gel)

Fig. 3. Absence of Pif1 results in fragmentation of mtDNA in the presence of ethidium bromide. Undigested total genomic DNA was purified from WT and pif1D mutant strains (treated or un-treated with 1 lg Et–Br/ml culture medium for 24 h) and DNA concentrations were determined by spectro-photometry. One microgram total DNA (undiluted) each were separated in twofold dilutions (1·, 2·, 4·, 8· concentrated) by agarose gel and transferred to a membrane. The membrane was sequentially hybridized with mtDNA fragments encoding a part of the cox1 gene (top panel), and with a part of the chromosomal CEN4 gene (middle panel). The Et–Br stained agarose gel is shown as loading control (bottom panel). As a molecular weight standard a ‘‘1-kb ladder’’ (Invitrogen) is shown.

Since Et–Br induces fragmentation of mtDNA, we hypothesize that Pif1p is required to maintain the stability of mtDNA in the presence of Et–Br. Therefore, we examined the integrity of mtDNA under the influence of Et– Br in WT and pif1D cells. We observed that the pif1D cells had about 60% less mtDNA compared to WT cells after 24 hrs of growth in the presence of Et–Br (Fig. 3, compare WT (+Et–Br) to pif1D (+Et–Br) strains). mtDNA in the pif1D strain treated with Et–Br was clearly fragmented in comparison to the mtDNA of the WT strain under similar conditions. The effect of Et–Br on mtDNA is specific, since the integrity of chromosomal DNA does not appear to be affected. As shown in the middle panel of Fig. 3, the amount and integrity of chromosomal DNA is comparable in WT and pif1D mutant cells independent of the presence or absence of Et–Br. In summary these data support the hypothesis that Pif1p is required to maintain mtDNA under conditions that introduce dsDNA breaks in mtDNA. Pif1p may have a role either in the prevention or repair of dsDNA breaks in mtDNA. 3.2. Absence of Pif1p results in specific mtDNA breakage We used the neutral-neutral two-dimensional (2D) gel electrophoresis technique to determine whether Pif1p affects replication and recombination of mtDNA (Brewer

215

and Fangman, 1987; Brewer et al., 1988). This technique separates branched DNA structures such as replication and recombination intermediates from linear non-replicated structures (Fig. 4B, cartoon). MtDNA from WT and pif1D strains was purified by CsCl gradient centrifugation to preserve low abundant replication intermediates. The mtDNA was digested with restriction endonucleases (BamHI or EcoRV) and the resulting DNA fragments were separated in two dimensions. In the first dimension the DNA fragments are separated according to molecular weight, in the second dimension the DNA intermediates are separated according to shape. Different shaped replication intermediates can therefore be separated from each other such as conventional replication forks (Fig. 4B, cartoon). Foury and colleagues showed that recombination between full-length and truncated versions of mtDNA is dependent on the presence of Pif1p in certain areas of mtDNA (Fig. 4A) (Foury and Kolodynski, 1983; Foury and Dyck, 1985). Therefore we examined whether Pif1p affects replication and recombination processes located in the ori6/var1 area (Fig. 4A, 5.9 kbp BamHI fragment). First, we noticed that pif1D cells showed on average about 50% fewer mtDNA replication intermediates compared to WT cells (Fig. 4B). These results suggest that the frequency of replication initiation is lower in mtDNA, or that replication forks are moving faster in the pif1D strains in the examined ori6/var1 area of mtDNA, or that replication intermediates are lost by mtDNA breakage. Given that recombination events within the examined area are dependent on Pif1p (Fig. 4A) (Foury and Kolodynski, 1983; Foury and Dyck, 1985), we assumed that mtDNA breakage events may occur that accompany the recombination activities in this area. Strikingly, several spots representing specific replication intermediates appear on the arc of linear fragments in the pif1D strain, but not in the WT strain. Based on our previous work with chromosomal replication we hypothesize that DNA breakage events may occur preferentially in front of replication forks that migrate through the examined ori6/var1 area (Ivessa et al., 2000; Ivessa et al., 2002; Ivessa et al., 2003). These breakage events create molecules that migrate to positions in 2D gels where linear DNA molecules are placed (arc of linear fragments; Fig. 4B, cartoon). In comparison, we also analyzed a different region of mtDNA in the WT and pif1D cells (Fig. 4A, EcoRV fragment within the cox1 gene) that is located outside of the Pif1-dependent recombination-area. When the replication pattern of the EcoRV fragment within the mitochondrial cox1 gene was examined by 2D gel technique, no significant differences in the replication pattern between WT and the pif1D cells were observed (Fig. 4B, right panels). These results suggest that Pif1p is required to prevent or repair mtDNA breakage at specific regions under conditions that do not include the exposure to a genotoxic chemical.

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X. Cheng et al. / Mitochondrion 7 (2007) 211–222 Pif1 - dependent recombination

A atp6 cox1 atp8

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Fig. 4. Absence of Pif1p causes specific breakage in mtDNA. (A) Schematic representation of mitochondrial DNA. Genetic elements are indicated by boxes or bars (Foury et al., 1998). Putative replication origins are indicated by grey, open circles. Individual tRNA genes are indicated with grey, vertical bars and labeled underneath with the letter ‘‘t’’, and rRNA genes are shown as black, horizontal bars. The sites where Pif1-dependent recombination occurs are indicated on top of panel A (Foury and Kolodynski, 1983; Foury and Dyck, 1985). The 5.9 kbp BamHI, and the 5.5 kbp EcoRV fragments, respectively that were analyzed by replication 2D gel technique are indicated in panel A at the bottom. (B) Replication intermediates of mtDNA were analyzed by 2D gel electrophoresis. As shown in the cartoon, structure [1] indicates non-replicating DNA hybridizing to a specific probe (1N spot). Position [2] indicates replication intermediates with fork-like structures. Position [3] indicates replication intermediates with two forks migrating in convergent orientation. These replication molecules migrate as a spike emanating from the 1N spot indicating that termination of replication may occur at this particular location. WT and pif1D cells were grown in glycerol-containing medium to an early logarithmic growth phase. mtDNA was purified by CsCl gradient centrifugation, digested with BamHI (A–D) or EcoRV (E and F). Hybridization was carried out using radiolabeled var1 (BamHI fragment; A–D) or cox1 (EcoRV fragment; E and F). For the BamHI fragment short (first row) and long (second row) exposures are shown. MtDNA breakage products are indicated with asterisks. DNA breakage likely occurs in front of replication forks (cartoon in the middle). The replication pattern of the mtDNA fragments that are recognized by the cox1 probe (E and F) is very similar in wild-type and pif1D samples.

3.3. Pif1p is associated with mtDNA Although Pif1p was first found to be localized to mitochondria (Lahaye et al., 1991), there is no evidence supporting an association of Pif1p with mtDNA. We used chromatin immunoprecipitation (ChIP), to test whether Pif1p binds to mtDNA in vivo at specific locations (Hecht and Grunstein, 1999; Hecht et al., 1999; Kuo and Allis, 1999). Since mtDNA is not assembled in a higher order molecular structure called ‘‘chromatin’’, we refer to the mtDNA immunoprecipitation technique as mIP. Pif1p may be specifically recruited or constitutively bound to specific sites in mtDNA to prevent or repair damage such as dsDNA breaks. Alternatively, Pif1p may migrate with the mitochondrial replication fork machinery. Using the mIP method we were able to detect whether Pif1p associates with specific sites of mtDNA or coats the entire mtDNA molecule. The mIP technique combines the immunoprecipitation technique and PCR (polymerase chain reaction). Form-

aldehyde is added to a growing yeast culture to establish cross-links between proteins and DNA and between proteins themselves. DNA with its proteins bound is sheared by sonication and the protein of interest (here: Pif1p) is precipitated using a specific antibody. The enrichment and specificity of protein bound to DNA is determined by PCR. We constructed a strain that places 13myc-epitopes in frame at the C-terminal end of the chromosomal-localized PIF1 gene to detect the presence of Pif1p in the cells (Longtine et al., 1998). The Pif1 protein migrates at the expected size of 114 kDa, the lane with the untagged control strain does not show any proteins that cross-react with the anti-myc antibodies (Fig. 5E). The strain expressing the myc-tagged Pif1 protein grows on Et–Br containing glycerol medium with the same rate as a WT strain expressing the untagged version of Pif1p suggesting that the myc-tag does not interfere with the function of the Pif1 protein (data not shown).

X. Cheng et al. / Mitochondrion 7 (2007) 211–222

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cob ori6 ori3 21S rRNA cox3ori5

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Input (PCR of mtDNA fragment)

Enrichment: (Pif1 / ut) ± Standard deviation

6.5 ± 0.71 4.6 ± 0.16 5.0 ± 0.14 4.6 ± 0.10 4.8 ± 0.51 6.8 ± 1.1 4.9 ± 0.12

Fig. 5. The DNA helicase Pif1p is associated with mtDNA in vivo. (A) Seven different DNA fragments were amplified from mtDNA by PCR. (B) Since pieces of mtDNA may also be found as inserts in chromosomal DNA, the primers for the PCR were tested whether they amplify specifically mtDNA within mitochondria. Two strains were compared: a strain that contains mtDNA (labeled as +) and a strain that lacks mtDNA (labeled as ). As a control, DNA fragments located within the chromosomal ARO1 (371 bp) gene were amplified by PCR. (C) A WT strain (ut for untagged) with the unmodified chromosomal PIF1 gene and a strain expressing a C-terminal myc-tagged version of the chromosomal PIF1 gene (Pif1) were used for the mIP experiment. The strain with the myc-tagged Pif1p expresses Pif1p at WT levels. Both strains were grown in rich (YEP) medium with glycerol as the carbon source to an early logarithmic growth phase. One part of the culture was treated with formaldehyde (cross-link) and the other part remained untreated (NO cross-link). All four samples were processed according to the mIP protocol. A monoclonal anti-myc antibody and Protein G coated magnetic beads were used to immunoprecipitate Pif1p. Lanes 1–8, PCR amplification products of the ori6 mtDNA fragment using DNA from the IP samples. Lanes 9–16, PCR amplification products of the ori6 mtDNA fragment using DNA from the input samples. The ‘‘input’’ samples represent the whole genomic DNA before the immunoprecipitation was carried out. The PCRs of nt-IP, Pif1-IP, and of the corresponding inputs were carried out in two-fold dilutions. Lane 1–6 and 9–14, formaldehyde cross-linked samples. Lane 7, 8, 15, and 16, Non-crosslinked samples. Most of the signal is reduced to background level in the non-crosslinked samples suggesting that the signals in lanes 4–6 (Pif1) are dependent on the cross-link. Pif1p is enriched about 5-fold over the nt control at the ori6 locus. (D) A similar experiment as described in C was repeated using the same strains as in (C) a WT strain (ut for untagged) with the unmodified chromosomal PIF1 gene and a strain expressing a C-terminal myc-tagged version of the chromosomal PIF1 gene (Pif1). Both strains were grown in rich (YEP) medium with glycerol as the carbon source to an early logarithmic growth phase. Top panel: PCR amplification products of the indicated mtDNA fragments using DNA from the IP samples. Bottom panel: PCR amplification products of the same indicated mtDNA fragments using DNA from the input samples. Below, the enrichment values (value from the Pif1 mIP versus value from the ut control) and the standard deviations from two independent experiments (two independent samples per experiment) are shown. Pif1p associates with several different locations in mtDNA. (E) Western blot of the IP samples using the anti-myc antibody confirming that Pif1 was efficiently precipitated from the crosslinked protein/DNA sample. No signal is visible in the untagged (ut) control.

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We first tested the specificity of the primers to amplify mtDNA (Figs. 5A and B). All primer sets could be used for the mIP, because they amplified mtDNA only from those samples that contained both chromosomal and mtDNA (Fig. 5B, lanes labeled as (+) mtDNA). If total genomic DNA was used that lacked mtDNA, the mtDNA-specific PCR products were absent (Fig. 5B, lanes labeled as () mtDNA). We used these primer pairs to perform the mIP experiment to assay for in vivo binding of Pif1p to mtDNA. The strain was used that expresses WT Pif1p as a C-terminal myc-tagged version. Pif1 protein is physically linked to mtDNA, if elevated amounts of PCR products are obtained using the strain with the tagged Pif1 protein version. In contrast, if a strain only expresses an untagged PIF1 gene, only background PCR signals are obtained. First, we tested whether a signal that is obtained with the myc-tagged Pif1p version is dependent on the cross-linker formaldehyde (Fig. 5C). We processed samples that were either treated or not treated with formaldehyde. PCR fragments were amplified that derive from a region in mtDNA that is directly adjacent to the ori6 origin. The enrichment of the ori6-PCR product derived from the strain containing the tagged Pif1p version is about 5-fold over the corresponding WT strain with the untagged Pif1p version (Fig. 5C, compare lane 1 with lane 6). In contrast, the samples that derived from the strains that were not treated with formaldehyde resulted only in a background PCR-signal that is comparable to the untagged Pif1p version (Fig. 5C, compare lane 1 with lane 8). These results suggest that the PCR signal derived from the ori6 mtDNA locus is only dependent on Pif1p that is physically attached to mtDNA before the yeast cells are lysed. This result excludes the possibility that Pif1p associates with mtDNA after lysis of the yeast cells. Next we tested whether Pif1-myc (Pif1) protein binds to other locations in mtDNA using the same primer pairs that were tested for mtDNA specificity (Figs. 5A and B). For comparison we also determined the binding behavior of the untagged Pif1 (ut) protein version to these sites in mtDNA (Fig. 5D). These regions are located within the Pif1-dependent and Pif1-independent recombination areas (Fig. 5A). They include the putative replication origins (ori3, ori5, and ori6), rRNA genes (21S rRNA), and mitochondrial genes that express proteins (cox1, cox3, and cob). When the signals derived from the tagged Pif1p version are compared to the untagged Pif1p versions, Pif1p shows binding to all seven examined sites in mtDNA. The enrichment of Pif1p to mtDNA (Pif1) was determined to be on average about 5.3-fold over the untagged control (ut). In general, these results suggest that Pif1p coats most likely the entire mtDNA molecule and binds to Pif1-dependent as well as Pif1-independent recombination areas. 4. Discussion The DNA helicase Pif1p is localized to two organelles: the nucleus and mitochondria (Lahaye et al., 1991; Zhou

et al., 2000). Previously, it was demonstrated that the nuclear form of Pif1p is attached to chromosomal DNA (Zhou et al., 2000). Here, we report for the first time that mitochondrial Pif1p is associated with mtDNA and therefore influences mtDNA function and this influence is likely direct. Furthermore, we show that yeast cells lacking mitochondrial Pif1p are hyper-sensitive to the genotoxic drug Et–Br (Figs. 1 and 2), and that their mtDNA is prone to fragmentation (Fig. 3). In contrast, WT cells grow normally under these conditions and their mtDNA is only slightly fragmented. Since Et–Br induces the formation of dsDNA breaks preferentially in mtDNA, we conclude from these experiments that mitochondrial Pif1p protects mtDNA from genotoxic stress. Using the replication 2D gel technique, we observe that mtDNA is breaking at specific sites in the absence of Pif1p already under natural growth conditions (Fig. 4, BamHI fragment). DNA breakage occurs in the same region of mtDNA that was previously shown to be affected by Pif1p in a recombination assay (Foury and Kolodynski, 1983; Foury and Dyck, 1985). Other regions in mtDNA, which are located outside of the Pif1-dependent recombination area, do not show any differences in the replication pattern between WT and pif1D strains (Fig. 4B, EcoRV fragment). Since breakage of mtDNA in the pif1D strain occurs at specific sites, replication intermediates are able to be visualized that have distinct sizes on the arc of linear fragments (Fig. 4B). If breakage occurs randomly in mtDNA, the broken mtDNA fragments would be distributed equally over the entire arc of linear fragments and sites of signal enrichment would be observed. We propose that breakage in mtDNA may occur in front of replication forks at particular breakage points (Ivessa et al., 2000; Ivessa et al., 2002; Ivessa et al., 2003). Since Pif1p is associated with the entire mitochondrial genome, as assayed by the mtDNA immunoprecipitation technique (Fig. 5), but lack of Pif1p only shows an effect on mtDNA replication locally, Pif1p may only be activated in certain regions of the mitochondrial genome. For example, Pif1p may associate with specific proteins in the active regions of mtDNA, or alternatively Pif1p could recognize certain DNA secondary structure in these regions that form during replication, repair or recombination. It was proposed previously by Foury’s laboratory that Pif1p recognizes specific recombinogenic signals in the cob/var1 and 21S rRNA regions of mtDNA. From these studies the authors concluded that Pif1p may act by recognizing topological structures instead of specific sequences (Foury and Dyck, 1985). We hypothesize that Pif1p either prevents or repairs breaks in mtDNA. Several studies regarding the nuclear function of Pif1p point to it’s role in the repair of dsDNA breaks. It’s known function in the nucleus suggests a likely function for Pif1p in the mitochondria. For example, nuclear Pif1p colocalizes with the homologous recombination protein Rad52p in the nucleus (Wagner et al., 2006). Rad52p typically exhibits diffuse nuclear localization but redistributes to discrete foci within the nucleus at sites of

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dsDNA breaks during S-phase. These sites are thought to act as repair centers for damaged DNA (Lisby et al., 2001; Lisby et al., 2003). If cells are exposed to ionizing radiation, dsDNA breaks occur and in response Rad52p focus formation is increased (Lisby et al., 2001). Similarly, the number of Pif1p foci is also increased in response to ionizing radiation treatment (Wagner et al., 2006). Further, PIF1 genetically interacts with the topoisomerase TOP3, and the DNA helicase SGS1, respectively (Wagner et al., 2006). Both TOP3 and SGS1 participate in the repair of chromosomal dsDNA breaks (Wallis et al., 1989; Gangloff et al., 1994; Chakraverty et al., 2001; Ira et al., 2003). We propose, therefore, that mitochondrial Pif1p may also participate in the repair of dsDNA breaks in a similar way as seen in the nucleus. It is possible that Pif1p is specifically targeted to dsDNA breaks in mtDNA to induce repair, most likely by homologous recombination. Since multiple identical copies of mtDNA are present in each cell a homologous repair template would be available. Additional support for the hypothesis that Pif1p has a role in DNA repair (perhaps dsDNA breaks) comes from the finding that PIF1 shows homology to the yeast RAD3 and E. coli RecD DNA helicases (Foury and Lahaye, 1987; Boule and Zakian, 2006). Rad3 is required for the excision repair of damaged DNA (Naumovski et al., 1985). RecD is a subunit of the RecBCD complex and is involved in the formation of 3 0 ssDNA tails, thereby initiating repair and recombination processes (Roman and Kowalczykowski, 1989). Both Rad3 and RecD unwind DNA with the same polarity as Pif1, 5 0 –3 0 . Furthermore, both RecD and Pif1p exhibit ssDNA-dependent ATPase activity (Dillingham et al., 2005). If the mitochondrial Pif1p participates in the formation of the 3 0 ssDNA overhang at dsDNA breakage sites to induce recombination and repair of mtDNA, it may facilitate this process in a similar way to RecD in bacteria. Several mitochondrial proteins are known to affect homologous recombination in mtDNA (Larsen et al., 2005) and these proteins may also be involved in the repair of dsDNA breaks in mtDNA. If Pif1p has a role in this type of repair in the mitochondria we may expect that Pif1p interacts with one or more of these proteins on a functional level. Some examples are the 5 0 –3 0 DNA exonucleases Nuc1p and Din7p that may process dsDNA breaks (Zassenhaus and Denniger, 1994; Fikus et al., 2000; Koprowski et al., 2003; Mookerjee and Sia, 2006). If Nuc1p and/or Din7p are involved in the creation of the 3 0 ssDNA overhangs at dsDNA breakage sites, Pif1p may facilitate the formation of these overhangs. Another example is the recombination protein Mhr1p that promotes strand-invasion of broken DNA ends into homologous DNA sequences (Ling et al., 2000; Ling and Shibata, 2002; Ling and Shibata, 2004; Mookerjee and Sia, 2006). Pif1p may facilitate this process by unwinding dsDNA where the 3 0 ssDNA strand is invading the duplex. Pif1p may also interact on a functional level with the cruciform cutting endonuclease Cce1p that resolves recombi-

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nation intermediates such as cross-overs (e.g. Hollidayjunctions) (Ezekiel and Zassenhaus, 1993; Lockshon et al., 1995). Pif1p may participate in the stabilization or resolution of recombination intermediates such as Holliday-junctions. Recombination occurs quite frequently in yeast mtDNA, as well as, in mammalian mtDNA (Lockshon et al., 1995; Kajander et al., 2001; Phadnis et al., 2005). Alternatively, Pif1p may prevent the formation of dsDNA breaks. For example, Pif1p may reduce the occurrence of deleterious DNA secondary structure. At elevated growth temperatures (36 C) pif1 mutant strains rapidly lose their mitochondrial genome (Van Dyck et al., 1992). This phenotype can be partially rescued by the over-expression of the single-strand binding protein Rim1. Since the mitochondrial genome is A/T rich (about 82%; (Foury et al., 1998)), yeast mtDNA may undergo local melting and form DNA secondary structures at elevated growth temperatures. In this model, Pif1p may migrate with the replication fork and help to resolve deleterious DNA secondary structures that otherwise may cause DNA breakage and consequently loss of mtDNA. Similar proteins exist in the nucleus, which are believed to unwind these types of secondary structures. One example is the Bloom’s syndrome DNA helicase, which can resolve G4 DNA (Watt and Hickson, 1996; Sun et al., 1998). Another example is the nuclear DNA helicase Srs2p that prevents the formation of deleterious recombination intermediates (Krejci et al., 2003; Veaute et al., 2003). Srs2p prevents binding of the recombination protein Rad51p to 3 0 ssDNA overhangs, thereby regulating the strand-invasion step that initiates the recombination process. We propose that mitochondrial Pif1p may also regulate recombination events in mtDNA as Srs2p does. If Pif1p is absent, the strand-invasion process may take place randomly, thereby increasing the probability of forming deleterious recombination intermediates and inducing dsDNA breaks in mtDNA. Pif1p may dislodge proteins from mtDNA either to control recombination events or just to facilitate replication fork progression. Pif1’s homology to Rrm3p supports this hypothesis (Ivessa et al., 2000). It is proposed that Rrm3p may temporarily dislodge non-histone binding proteins from chromosomal DNA to facilitate replication fork progression past specific sites such as centromeres, tRNA and rRNA genes, or silent replication origins (Ivessa et al., 2003; Azvolinsky et al., 2006). Recent results suggest that mitochondrial Pif1p may have additional roles in mitochondrial recombination (O’Rourke et al., 2002). Lack of Pif1p exhibits a synthetic petite-phenotype in combination with ntg1 null mutations that is accompanied by enhanced mtDNA point mutagenesis in the corresponding double-mutant strain (O’Rourke et al., 2002). Ntg1 is a member of the mitochondrial base excision repair pathway and removes oxidized pyrimidines as a DNA glycosylase (Alseth et al., 1999; You et al., 1999). However, since lack of other mitochondrial recombination proteins such as the cruciform cutting endonuclease Cce1p and the nuclease Nuc1p do not display a synthetic-petite

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phenotype with ntg1 null mutants, and even the triple mutant cce1D nuc1D ntg1D exhibits only a mild synthetic effect on petite-mutant induction, Pif1p may have additional roles in mitochondria other than recombination (O’Rourke et al., 2002). The authors speculate that Pif1p may influence the structure or accessibility of mtDNA in a manner that facilitates binding or improves the activity of Ntg1p or other repair proteins. This may be a general function such as packaging of the mtDNA into nucleoids or promoting a specific DNA or protein/DNA conformation at sites of DNA damage. Interestingly, the respiratory-deficiency phenotype of a strain lacking Pif1p can be partially rescued by over-expression of a large subunit of ribonucleotide reductase, Rnr1p, which is the rate-limiting enzyme in dNTP synthesis (O’Rourke et al., 2005). In this way, the cellular dNTP synthesis is increased due to the increase in Rnr1p (Zhao et al., 1998). This observation seems to reflect the general need of regulated dNTP levels in ensuring optimal replication and repair activities in mitochondria since mutations in the only mitochondrial DNA polymerase MIP1 can be rescued by altering the dNTP pool levels (Lecrenier and Foury, 1995; Zhao et al., 2001). Therefore, it might be interesting to know if over-expression of Rnr1p also partially suppresses the Et– Br sensitivity and/or the replication defects observed in the pif1D strain. This result may suggest that nucleotide amounts may also be a determining factor in the prevention or repair of dsDNA breaks. Previously, it was suggested that an imbalance in the nucleotide pool within mitochondria may lead to replication fork stalling and dsDNA breaks in mtDNA (Nishigaki et al., 2003; Song et al., 2003). Future studies should demonstrate if mitochondrial Pif1p is needed either to prevent or repair damaged mtDNA (ex. dsDNA breaks). Acknowledgements We are grateful to Drs. Ronald Butow, Donna Gordon, Maria Mateyak, Debkumar Pain, and Carolyn Suzuki for discussions and providing reagents. This work is supported by a startup grant from the Department of Cell Biology and Molecular Medicine of the University of Medicine and Dentistry NJ, by a grant from the Foundation of the University of Medicine and Dentistry, NJ, and by a grant from the New Jersey Commission on Cancer Research (05-1975-CCR-EO). References Alseth, I., Eide, L., Pirovano, M., Rognes, T., Seeberg, E., Bjoras, M., 1999. The Saccharomyces cerevisiae homologues of endonuclease III from Escherichia coli, Ntg1 and Ntg2, are both required for efficient repair of spontaneous and induced oxidative DNA damage in yeast. Mol. Cell. Biol. 19, 3779–3787. Azvolinsky, A., Dunaway, S., Torres, J.Z., Bessler, J.B., Zakian, V.A., 2006. The S. cerevisiae Rrm3p DNA helicase moves with the replication fork and affects replication of all yeast chromosomes. Genes Dev. 20, 3104–3116.

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