The mitochondrion has been an identified subcellular organelle since the late 1800s, though its function and importance remained relatively cryptic until almost a century later. With the molecular renaissance of the 1950s and 1960s came the tools and perspectives, including peptide sequencing, microscopy, and the chemiosmotic hypothesis, with which to formulate and test crucial questions about what mitochondria do and how they function. In addition, shortly after the structure of DNA was elucidated and its role as the genetic material of the cell established, the presence of extranuclear DNA was revealed by electron microscopy (Nass and Nass, 1963) and by density gradient fractionation (Haslbrunner et al., 1964), identifying this DNA as part of highly purified mitochondria.
The goal of targeted genetic manipulation within the mitochondrial genome is rapidly driving a second renaissance in mitochondrial biology. Current approaches to the mitochondrial uptake of DNA have recently been reviewed in detail (Mileshina et al., 2011a). Several model organisms are currently amenable to this technique although no vertebrate species are among them. In our view, successful mitochondrial transformation is hindered by our limited knowledge of the fundamental processes of mitochondrial DNA maintenance and repair. We are hopeful that a greater understanding of the replication, repair, and maintenance of the mitochondrial genome, afforded by the tools currently in existence and presented here, will soon allow the construction of mammalian mitochondrial disease models (Dunn et al., 2011), or lead directly to gene therapy for the treatment of mitochondrial diseases in patients (Schon and Gilkerson, 2010). In this review, we will discuss the current state of mitochondrial genetic reporters and their application toward understanding and manipulating mitochondrial genome maintenance.
In cells, mitochondrial DNA is organized into protein-associated structures called nucleoids. Quantitative PCR coupled with immunofluorescence microscopy revealed an estimated 2-8 mitochondrial genomes per nucleoid in human immortalized cell culture (Legros et al., 2004); higher-resolution microscopy identified more nucleoids per cell, bringing this estimate down to only 1-2 copies per nucleoid (Kukat et al., 2011). A growing number of the proteins associated with the mitochondrial nucleoid have been identified from eukaryotic model systems (Bogenhagen et al., 2003; Garrido et al., 2003; Kienhöfer et al., 2009). The conserved and abundant HMG protein, Abf2p in yeast, and mtTFA (or TFAM) in vertebrates, is thought to organize and compact the mitochondrial genome, and is proposed to play a histone-like role in mitochondrial DNA organization (Kaufman et al., 2007; Pohjoismaki et al., 2006). In addition to these properties, TFAM has recently been reported to contribute to mitochondrial DNA replication (Pohjoismaki et al., 2006) and repair (Canugovi et al., 2010).
The mitochondrial genome is vitally important to the organelle’s proper function. Its encoded proteins are almost all subunits of the mitochondrial respiratory complexes; mutations to these genes can disrupt bioenergetic function and control. There is also growing evidence that proper maintenance of the mitochondrial genome is tightly associated with normal mitochondrial behavior, including fusion, fission, and intracellular migration (Baker and Haynes, 2011; Chen et al., 2010; Gilkerson, 2009). The reasons for this are currently unknown, but we can speculate that nucleoids may help to define a minimal mitochondrial “unit” and that disruption of mitochondrial DNA resolution affects mitochondrial dynamics and transmission in dividing cells (Margineantu et al., 2002). Alternately, nucleoids may help to catalyze organization of cristae, respiratory complexes, or other submitochondrial structures, as has been suggested for the Complex V ATP synthase (Strauss et al., 2008), and faulty mitochondrial DNA maintenance disrupts this patterning.
Fission and fusion, coupled with autophagic degradation of mitochondrial material (“mitophagy”), are increasingly appreciated as a primary means of mitochondrial quality control. Instability of the mitochondrial genome is therefore not just a problem affecting its encoded products, but potentially the structural integrity of the entire organelle. Recent proposals suggest that the cellular dysfunction underlying neurodegeneration in Alzheimer’s and Parkinson’s diseases is caused or at least exacerbated by defective mitophagy and increased mitochondrial DNA instability (Chang, 2000; Corral-Debrinski et al., 1994; Coskun et al., 2011; Narendra et al., 2010; Narendra et al., 2008; Sasaki et al., 1998; Suen et al., 2010).
1.1. Yeast gene nomenclature
A comprehensive guide to yeast gene nomenclature is both published (
There are two nomenclature systems, reflecting the advent of genomic analysis. The ORF naming system, instituted in the post-genomic era, gives each predicted open reading frame a systematic designation reflecting its chromosomal position and strand orientation. This system will not be discussed here. We will focus on gene symbol nomenclature, which applies only to ORFs that express a known gene product and is in common use for most proteins.
In gene symbol nomenclature, yeast genes are given a three-letter “name” followed by a number. Gene names are italicized, with dominant (usually wild-type) alleles in capital and recessive (usually mutant) alleles in lowercase type. For example,
Mitochondrial dysfunction can occur through single gene mutations in both nuclear and mitochondrial genomes, forming two classes of mutants.
Generation of mitochondrial DNA-free derivatives of any strain is achieved by culturing cells wtith ethidium bromide, which blocks mitochondrial DNA replication without affecting nuclear DNA (Meyer and Simpson, 1969 ). These ρ0 strains can be studied directly or used as a tool for mitochondrial DNA manipulation.
1.2. The presence of mitochondrial DNA repair mechanisms
The assertion that mitochondrial DNA did not undergo repair was made as late as 1990 (Singh and Maniccia-Bozzo, 1990), in agreement with early reports (Clayton et al., 1974). Part of the appeal of this idea was the observation that mitochondrial DNA depletion can be induced by various insults, including oxidative stress (Shokolenko et al., 2009), ethanol (Ibeas and Jimenez, 1997) and zidovudine (AZT) (Arnaudo et al., 1991) treatment. This was interpreted as evidence that, when damaged, mitochondrial DNA was simply eliminated rather than repaired. However, in 1992, the first evidence for photolyase repair of UV-induced mitochondrial DNA damage in yeast was provided (Yasui et al., 1992), followed quickly by work from the Bohr and Campbell groups demonstrating uncharacterized repair activities and homologous recombination, respectively, in two mammalian mitochondrial systems (LeDoux et al., 1992; Thyagarajan et al., 1996). The fifteen years since have revealed an extensive set of mitochondrial DNA repair pathways, including base excision repair (BER), homologous recombination (HR), and non-homologous end joining (NHEJ). A recent review summarizes our current understanding of these pathways in mitochondria and other organellar genomes (Boesch et al., 2010).
1.3. Nuclear and mitochondrial DNA repair pathways share protein components
Many of the known mitochondrial DNA repair pathway proteins are mitochondrially-localized proteins initially characterized in nuclear repair. The first mitochondrial DNA repair protein identified, photolyase, was demonstrated to be one such dual-localized protein (Green and MacQuillan, 1980). Subsequent studies indicated localization of base excision repair proteins to both subcellular compartments, including the yeast glycosylases Ntg1p (You et al., 1999), Ung1p (Chatterjee and Singh, 2001) and Ogg1p (Singh et al., 2001), the mammalian glycosylases UNG1 (Nilsen et al., 1997), MTH1 (Kang et al., 1995), OGG1 (Nishioka et al., 1999), and MYH (De Souza-Pinto et al., 2009; Nakabeppu et al., 2006), and the yeast AP endonuclease Apn1p (Ramotar et al., 1993). In human lymphoblasts, BER proteins were associated with the mitochondrial inner membrane fraction, where mitochondrial nucleoids are also found (Stuart and Brown, 2006). These findings illustrate the high evolutionary conservation of mitochondrial BER. Factors that regulate the subcellular localization of these proteins are not well understood; however, changes to localization in response to stress has recently been demonstrated (Griffiths et al., 2009; Swartzlander et al., 2010). This apparent recruitment of DNA repair proteins to the mitochondria may represent a DNA-specific communication pathway between the intramitochondrial and extramitochondrial environments.
Other DNA repair proteins have been shown to affect mitochondrial DNA maintenance in mammalian cells, including the BER flap endonuclease FEN1 (Liu et al., 2008), DNA double-strand break repair proteins, Rad51p (Sage et al., 2010), Mre11 (Dmitrieva et al., 2011) and Ku80 (Coffey et al., 1999), and the nucleotide excision repair protein CSA (Kamenisch et al., 2010). In addition, in yeast, DNA damage tolerance pathways that utilize the translesion polymerase complexes encoded by Rev1p, Rev3p, and Rev7p also impact mitochondrial mutagenesis (Kalifa and Sia, 2007; Zhang et al., 2006).
2. Manipulation of the yeast mitochondrial genome
2.1. Basic features of the mitochondrial genome
The yeast mitochondrial genome consists of 75-85 kb of double-stranded DNA, encoding seven protein products (Cox I, II, III, Atpase 6, 9, cyt b, Var1), 2 rRNAs and 24 tRNAs, while the human mitochondrial genome is a much smaller 16.5 kb and encodes 13 protein products (cox I, II, III, ND1-6, 4L, Atpase 6, 8, cyt b), 2 rRNAs and 22 tRNAs. Aside from the presence of complex I (NADH:ubiquinone oxidoreductase) subunit genes in the human mitochondrial genome and not yeast, and the presence of non-coding regions in the yeast genome, the two are remarkably similar in structure and encoded products, giving yeast mitochondrial genome manipulation great power to inform our understanding of mammalian mitochondrial DNA defects.
Mitochondrial and nuclear DNA in yeast are compositionally different; mitochondrial DNA is relatively AT-rich and highly repetitive, with G and C bases further segregated in coding regions. This repetition made initial sequencing of the entire yeast mitochondrial genome difficult (Foury et al., 1998) and is a continued challenge in targeted gene manipulation, particularly in the intergenic AT-rich regions. Yeast mitochondrial DNA has multiple regions of non-coding DNA, which are the primary contributors to the 83% AT bias of the genome. The size difference between human and yeast mitchondrial DNA is almost entirely due to the absence of these intergenic regions in the human mitochondrial genome.
2.2. Organisms with tractable mitochondrial genomes
To date, the number of organisms that have successfully undergone mitochondrial transformation remains small and is restricted to unicellular eukaryotes. The most widely used model remains the budding yeast
The mitochondria of hyphal yeast
An important requirement of successful mitochondrial manipulation is the ability to select and purify a rare transformation event; in
The only known algal species to date that can be biolistically transformed is the green alga
Biolistic transformation in multicellular organisms is hampered by an inability to select and amplify individual transformants. A promising recent attempt to transform mitochondria in a mouse embryonic fibroblast line with a “universal” neomycin marker via a bacterial conjugation-like mechanism was not successful in generating mitochondrial transformants (Yoon and Koob, 2011). However, mitochondria isolated from both mammalian and plant sources have been successfully transformed
2.3. Biolistic transformation of yeast and selection of transformed clones
Microprojectile bombardment of DNA on a carrier is an effective method for delivering DNA past the plasma membrane and two mitochondrial membranes into the mitochondrial matrix. This method was pioneered in plants by John Sanford (Sanford et al., 1987), and first demonstrated in yeast by Sanford, Butow and colleagues (Johnston et al., 1988). Described below is the general transformation procedure used for
The linear or circular plasmid DNA to be transformed is alcohol-precipitated onto a carrier substrate, usually tungsten or gold particles <1µm. The bombardment itself occurs in a biolistic gun chamber (Sanford, 1988), where rising vacuum pressure ruptures a pressure sensitive disk holding the DNA-precipitated particles, driving the particles onto a freshly plated lawn of haploid yeast cells. The plate medium then selects for uptake of either the plasmid of interest or of a co-transformed marker if the target plasmid does not confer a selectable phenotype.
The target cells for mitochondrial transformation are typically ρ0 (non-mitochondrial DNA-containing) derivatives of a chosen strain, to ensure that the only mitochondrial DNA present is transformation-derived. After selection for the co-transformed nuclear marker, transformants must be screened for the presence of the transforming mitochondrial DNA. Following mitochondrial uptake of the desired DNA, positive haploid clones are mated to a strain containing wild-type mitochondrial genomes, allowing mixing of the transformed DNA with the target mitchondrial DNA. Generally, the desired outcome is integration of the synthetic DNA construct into the mitochondrial genome, although mitochondrial plasmid maintenance can also occur. The specific example of
ARG8 m auxotrophic mitochondrial reporter gene
3.1. Building an auxotrophic mitochondrial reporter: the
ARG8 m gene
The phenotypic output of a genetic reporter system determines its strengths and weaknesses as an analytic tool. In yeast, multiple auxotrophic (factor-requiring) mutants have historically been used with great success as both selective markers and phenotypic reporters. The defined requirements of yeast grown in culture allow for synthetic reconstitution of growth media lacking a specific amino acid. Commonly used auxotrophic markers in yeast include growth status on media lacking uracil, histidine, leucine, arginine, methionine, and lysine. Many laboratory strains, including our wild-type strain, DFS188 and its derivatives, lack the ability to make these amino acids due to specific nuclear mutations. These strains are typically maintained in rich media, allowing unrestricted growth. Withdrawal of the amino acid in question results in cell death. Rescue of a growth phenotype occurs when the gene that complements the nuclear mutation for that amino acid’s synthesis is supplied on a plasmid or as part of a conditional reversion construct, allowing cells to regain prototrophic (factor-independent) growth.
While an ideal system to assess mechanisms associated with nuclear gene expression, the mitochondrial genome has long been inaccessible to auxotrophic reporter manipulation because it does not encode any amino acid biosynthetic enzymes. Direct insertion of a nuclear gene is impossible, as the codon usage of the mitochondrial genome differs from the nuclear genome both in the preferred codon frequency and in some codon products. Multiple nuclear leucine codons encode threonine in mitochondria, and a nuclear UGA stop codon encodes tryptophan in the mitochondria of yeast (Bonitz et al., 1980). Generating a mitochondrial auxotrophic reporter thus requires mutating a nuclear gene to enable its expression from the mitochondrial genome. Once constructed, this gene must be introduced into the mitochondrial genome with the appropriate transcriptional and translational cues. To date, the only such auxotrophic marker gene to be engineered in this way is the synthetic
Fox and group began by synthetically generating a 1.3 kilobase fragment encoding the entire 423 amino acid acetylornithine transaminase enzyme. Substitutions were made at 12 CUN codons (n: Leu; mt: Thr) and 6 AUA codons (n: Ile; mt: Met) to maintain the Leu and Ile residues. In addition, each of the two Trp codons was changed to UGA (n: STOP; mt: Trp) ensuring
Several steps were then required to generate the desired end product of the
The plasmid containing
To allow mixing of the mitochondrial plasmid DNA with intact mitochondrial genomes, the biolistically transformed cells were mated with a second haploid strain bearing normal mitochondrial DNA (Fig. 1, Fig. 2A). Since one strain is karyogamy-deficient, the nuclear envelopes do not fuse. Cell division gives rise to haploid cells, and one haploid genome can be selected in subsequent divisions. The mitochondria, however, undergo rapid fusion, allowing interaction between plasmid and mitochondrial DNA. This process is known as
cytoduction. Homologous recombination between the
Mitochondrial genome incorporation of
ARG8 m as a reporter of mitochondrial translation, DNA repair, recombination, and heteroplasmy
Prior to the construction of the
colonies. Though petite formation is a somewhat useful indicator of gross mitochondrial DNA abnormality, its pleiotropic nature (the petite phenotype may result from nuclear
4. Measuring mitochondrial microsatellite instability
Short, repetitive sequences consisting of di- and tri-nucleotide repeats are abundant in the nuclear genome, in both coding and non-coding regions. Their appearance and inherent instability in the coding regions of several proteins is the underlying cause of the polyglutamine diseases, including Huntington’s disease and multiple types of spinocerebellar ataxia. These microsatellites are an important source of mutation in nuclear DNA through the internal repetition that facilitates repeat length changes by polymerase slippage, and through the ability of separate repeated regions to undergo homologous recombination in
Yeast mitochondrial DNA is highly microsatellite-enriched, partially due to the abundance (83%) of A and T. Mammalian mitochondrial DNA lacks most of the non-coding AT-rich DNA (~44% A/T) that gives rise to this bias, but still contains AT-rich repetitive regions (Anderson et al., 1980). If this repetition confers higher mitochondrial DNA instability, similar to its effects on nuclear DNA (Wierdl et al., 1997), it could play an important role in mitochondrial dysfunction as it relates to aging and aging-related diseases. These findings provided the impetus for making a mitochondrial microsatellite reporter system.
In 2000, Petes, Fox, and colleagues published an analysis of mitochondrial DNA microsatellite instability using the
The experiments carried out with these strains were among the first to demonstrate fundamental differences between nuclear and mitochondrial DNA processing and maintenance. Unlike nuclear DNA, in which poly(AT) and poly(GT) tracts have similar levels of instability, with repeat addition favored, mitochondrial poly(AT) tracts are much more stable than poly(GT) tracts, and repeat deletion predominates. These and other differences suggest that assumptions of mitochondrial DNA behavior based on nuclear DNA may be inherently flawed, preventing a clear understanding of how to predictably manipulate mitochondrial DNA.
To allow microsatellite instability measurement in respiring cells, a state that imposes a respiration requirement on mitochondrial DNA maintenance, we developed and characterized a version of the microsatellite reporter that is respiration competent (Kalifa and Sia, 2007; Mookerjee and Sia, 2006). This ensures that the measured microsatellite instability occurs in an otherwise functional background. This new reporter serves two useful purposes, allowing us to determine the effect of an active respiratory chain on mitochondrial mutagenesis, and to assess microsatellite instability in mutant strains that only maintain mitochondrial DNA under constant respiratory selection. For this reporter, instead of replacing
5. Measuring direct repeat-mediated deletions
Accumulation of mitochondrial deletions is associated with multiple pathologies and with aging. These deletions are commonly flanked by direct repeats, raising speculation that they are recombination-mediated. A detailed understanding of mitochondrial DNA recombination has lagged far behind equivalent nuclear recombination processes, which in turn limits our efforts to use recombination both as a molecular tool for genome manipulation and as a biological correlate of mitochondrial dysfunction. By manipulating the sequence context of the
Rep96::ARG8 m ::cox2’ reporter and variants
We generated a synthetic deletion substrate with the
Yeast bearing the
5.2. Characterizing direct-repeat mediated deletion (DRMD) in yeast
Work by Phadnis et al. (2005) demonstrates the utility of this reporter in characterizing mitochondrial recombination. First, generation of deletion reporters containing different repeat lengths revealed that the rate of
Second, the effects of heterology on deletion efficiency were tested by introducing silent mutations into either the leading or following repeat (relative to the direction of transcription), giving rise to ~2% heterology between the repeat sequences. This design was meant to allow comparison to similar work in the yeast nuclear genome, where a 3% heterology between 205 bp repeats decreased the rate of deletion formation 6-fold (Sugawara et al., 2004) These experiments revealed similar behavior in mitochondrial DNA, where a 3- to 4-fold reduction in deletion formation rate was observed. Interestingly, this effect was dependent on mutation placement in the leading repeat; the same mutations in the following repeat had no effect on deletion rate. One explanation for this is that the heteroduplex rejection mechanisms may act more stringently on particular mispair orientations. In successful deletion events, the final sequence was almost always that of the repeat closest to the remaining
The possible mechanisms mediating repeat-dependent deletion can be partially distinguished based on the DNA products they generate, namely, whether detectable products are reciprocal or non-reciprocal. Unlike qPCR detection methods that are commonly used to measure mitochondrial DNA deletions
Third, genes believed to be involved in repeat-mediated deletion were tested to determine their effects on the rate of mutation. Mutations to the proof-reading domain of the mitochondrial DNA polymerase were shown to affect
5.3. Assaying heteroplasmy in yeast
As stated earlier, the
6. Elucidation of mitochondrial DNA repair pathways
With specific reporters of microsatellite instability, point mutation, and direct repeat instability, coupled with direct sequencing and Southern blotting, the pathways of mitochondrial DNA repair become more readily accessible to quantitative analysis. This section will discuss some of the research findings resulting from use of the mitochondrial reporters described above.
6.1. Mismatch recognition combines with recombination and base excision repair pathways
Use of the
Mismatch repair has been a predicted pathway of mitochondrial DNA repair since the identification of the mitochondrially-localized MutS homolog, Msh1p. However, there is currently no direct evidence in yeast for mismatch repair activity. Human mitochondria do have a putative mismatch repair mechanism (De Souza-Pinto et al., 2009), but do not possess a MutS homolog. Point mutation accumulation rates increase with Msh1p disruption, but evidence from multiple groups suggests that this is due to base excision repair (BER), rather than mismatch repair (MMR), defects. Further, no other characterized mismatch repair proteins are known to localize to the mitochondria.
Haploid yeast strains with deletions of the
By comparison with the known mutations, all three
We then characterized the effects of
Mismatch repair proteins have been shown to function in other DNA repair pathways, including BER, nucleotide excision repair (NER), and homologous recombination (Goldfarb and Alani, 2005; Polosina and Cupples, 2010). Using the mitochondrial reporters, we were able to examine the genetic interaction of
Though widely accepted as a functional mechanism in mitochondrial DNA, the proteins that carry out recombination, and the specific mechanisms themselves, are largely unknown. Due to differences in the available proteins, in the substrate, or in the presumably constant availability of a homologous template, mitochondria may combine existing repair components is ways not seen in the nucleus (Masuda et al., 2009). We speculated that Msh1p, like its nuclear homologs, may play a role in the generation of deletions at directly-repeated sequences, and therefore would be predicted to result in reduced DRMD. Unexpectedly, we found that all three
Previously, examination of the deletion junctions of ρ- genomes in spontaneous
If Msh1p mutation allows it to bind but not release mismatched DNA, it is tempting to speculate that DNA molecules might become aberrantly linked. While not direct confirmation of this, we did find that the
In addition to suppressing DRMD, Msh1p is also an important component of mitochondrial base excision repair. Through conventional epistasis analysis of
6.2. Translesion polymerases facilitate frameshifts but suppress mitochondrial point mutation
The high fidelity of replicative DNA polymerases arises through extremely stringent requirements for nucleotide binding in the active site. While normally desirable, the inflexibility of these domains to accept alternate substrates causes replication fork stalling or collapse in the presence of cyclobutane-pyrimidine dimers, a common UV-induced product, and other damage resulting in large adducts.
To remedy this, a second class of polymerases exists that have much less stringent binding requirements for nucleotide incorporation. These translesion polymerases sacrifice fidelity for greater flexibility in template usage, favoring processivity but leading to mutagenic DNA synthesis that introduces both point and frameshift mutations. Consequently, their disruption gives rise to higher sensitivity to DNA damaging agents (e.g., UV) but lower nuclear DNA mutation rates in surviving cells.
Polγ is the only known mitochondrial replicative polymerase and, until recently, the only polymerase with known mitochondrial DNA activity. In yeast,
Using the respiring (GT16+1) and (GT16+2) reporters (Fig 2C), (Mookerjee and Sia, 2006), we found that single deletions of
These observations emphasize the importance of examining the effect of disrupting repair or damage tolerance pathways on multiple types of mutations, as they are often generated and repaired via distinct mechanisms. The employment of multiple mutagenesis reporters allows the proper dissection of these pathways.
Increasingly, proteins previously considered to be nuclear DNA repair factors are found to also display mitochondrial localization (Section 1.3), suggesting significant overlap between nuclear and mitochondrial DNA repair pathways with respect to their protein components. However, our analysis of the phenotypic consequences of mutating the relevant genes reveals significant differences in the contribution of these proteins to mitochondrial mutagenesis and repair (Sections 5 and 6). These differences likely result from compositional DNA differences, the different packaging and DNA-binding proteins, the different regulatory control of mitochondrial DNA replication and transmission, the exposure to certain kinds of damage, and the availability of other repair proteins, between nuclear and mitochondrial DNA. We should not expect that studies of nuclear DNA repair can simply be extrapolated to generate correct mitochondrial models.
Careful analysis of these pathways within the mitochondrion will require tools like those we have described here. These reporters provide us with the ability to differentiate between point substitutions, frameshift mutations, and deletion events and will be critical to elucidating specific pathways. While
Work described in this chapter was supported by the National Institutes of Health grant GM63626 and the National Science Foundation grants MCB0543084 and MCB0841857 to E. A. S.
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