Physiological and biochemical defects in functional interactions of mitochondrial DNA polymerase and DNA-binding mutants of single-stranded DNA-binding protein

Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos greatly enhance the overall activity of pol gamma by increasing primer recognition and binding and stimulating the rate of initiation of DNA strands (Farr, C. L., Wang, Y., and Kaguni, L. S. (1999) J. Biol. Chem. 274, 14779-14785). We show here that DNA-binding mutants of mtSSB are defective in stimulation of DNA synthesis by pol gamma. RNAi knock-down of mtSSB reduces expression to <5% of its normal level in Schneider cells, resulting in growth defects and in the depletion of mitochondrial DNA (mtDNA). Overexpression of mtSSB restores cell growth rate and the copy number of mtDNA, whereas overexpression of a DNA-binding and functionally impaired form of mtSSB neither rescues the cell growth defect nor the mtDNA depletion phenotype. Further development of Drosophila animal models, in which induced mtDNA depletion is manipulated by controlling exogenous expression of wild-type or mutant forms, will offer new insight into the mechanism and progression of human mtDNA depletion syndromes and possible intervention schemes.     

Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells

The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase γ (pol γ) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol γ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol γ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol γ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.     

Arabidopsis mitochondrial single-stranded DNA-binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination

Faithful DNA replication is one of the most essential processes in almost all living organisms. However, the proteins responsible for organellar DNA replication are still largely unknown in plants. Here, we show that the two mitochondrion-targeted single-stranded DNA-binding (SSB) proteins SSB1 and SSB2 directly interact with each other and act as key factors for mitochondrial DNA (mtDNA) maintenance, as their single or double loss-of-function mutants exhibit severe germination delay and growth retardation. The mtDNA levels in mutants lacking SSB1 and/or SSB2 function were two- to four-fold higher than in the wild-type (WT), revealing a negative role for SSB1/2 in regulating mtDNA replication. Genetic analysis indicated that SSB1 functions upstream of mitochondrial DNA POLYMERASE IA (POLIA) or POLIB in mtDNA replication, as mutation in either gene restored the high mtDNA copy number of the ssb1-1 mutant back to WT levels. In addition, SSB1 and SSB2 also participate in mitochondrial genome maintenance by influencing mtDNA homologous recombination (HR). Additional genetic analysis suggested that SSB1 functions upstream of ORGANELLAR SINGLE-STRANDED DNA-BINDING PROTEIN1 (OSB1) during mtDNA replication, while SSB1 may act downstream of OSB1 and MUTS HOMOLOG1 for mtDNA HR. Overall, our results yield new insights into the roles of the plant mitochondrion-targeted SSB proteins and OSB1 in maintaining mtDNA stability via affecting DNA replication and DNA HR.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein

Biochemical studies of the mitochondrial DNA (mtDNA) replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence) are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold). Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.     

Arabidopsis thaliana organellar DNA polymerase IB mutants exhibit reduced mtDNA levels with a decrease in mitochondrial area density

Plant organelle genomes are complex and the mechanisms for their replication and maintenance remain unclear. Arabidopsis thaliana has two DNA polymerase genes, DNA polymerase IA (polIA) and polIB, that are dual targeted to mitochondria and chloroplasts and are differentially expressed in primary plant tissues. PolIB gene expression occurs at higher levels in tissues not primary for photosynthesis. Arabidopsis T‐DNA polIB mutants have a 30% reduction in relative mitochondrial DNA (mtDNA) levels, but also exhibit a 70% increase in polIA gene expression. The polIB mutant shows an increase in mitochondrial numbers but a significant decrease in mitochondrial area density within the hypocotyl epidermis, shoot apex and root tips. Chloroplast numbers are not significantly different in mesophyll protoplasts. These mutants do not have a significant difference in total dark mitorespiration levels but exhibit a difference in light respiration levels and photosynthesis capacity. Organelle‐encoded genes for components of respiration and photosynthesis are upregulated in polIB mutants. The mutants exhibited slow growth in conjunction with a decreased rate of cell expansion and other secondary phenotypic effects. Evidence suggests that early plastid development and DNA levels are directly affected by a polIB mutation but are resolved to wild‐type levels over time. However, mitochondria numbers and DNA levels never reach wild‐type levels in the polIB mutant. We propose that both polIA and polIB are required for mtDNA replication. The results suggest that polIB mutants undergo an adjustment in cell homeostasis, enabling them to maintain functional mitochondria at the cost of normal cell expansion and plant growth.     

Mitochondrial dysfunction in cancer cells due to aberrant mitochondrial replication

Warburg effect is a hallmark of cancer manifested by continuous prevalence of glycolysis and dysregulation of oxidative metabolism. Glycolysis provides survival advantage to cancer cells. To investigate molecular mechanisms underlying the Warburg effect, we first compared oxygen consumption among hFOB osteoblasts, benign osteosarcoma cells, Saos2, and aggressive osteosarcoma cells, 143B. We demonstrate that, as both proliferation and invasiveness increase in osteosarcoma, cells utilize significantly less oxygen. We proceeded to evaluate mitochondrial morphology and function. Electron microscopy showed that in 143B cells, mitochondria are enlarged and increase in number. Quantitative PCR revealed an increase in mtDNA in 143B cells when compared with hFOB and Saos2 cells. Gene expression studies showed that mitochondrial single-strand DNA-binding protein (mtSSB), a key catalyst of mitochondrial replication, was significantly up-regulated in 143B cells. In addition, increased levels of the mitochondrial respiratory complexes were accompanied by significant reduction of their activities. These changes indicate hyperactive mitochondrial replication in 143B cells. Forced overexpression of mtSSB in Saos2 cells caused an increase in mtDNA and a decrease in oxygen consumption. In contrast, knockdown of mtSSB in 143B cells was accompanied by a decrease in mtDNA, increase in oxygen consumption, and retardation of cell growth in vitro and in vivo. In summary, we have found that mitochondrial dysfunction in cancer cells correlates with abnormally increased mitochondrial replication, which according to our gain- and loss-of-function experiments, may be due to overexpression of mtSSB. Our study provides insight into mechanisms of mitochondrial dysfunction in cancer and may offer potential therapeutic targets.     

Uncoupling the roles of the SUV3 helicase in maintenance of mitochondrial genome stability and RNA degradation

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.     

Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication

Mitochondrial DNA (mtDNA) is organized in discrete protein–DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.     

A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial DNA replication and polymerase activity

We have isolated a thermosensitive mutant which is transformed into a population of cells devoid of mitochondrial DNA (rho 0 cells) at 35 degrees C and is deficient in mitochondrial (mt) DNA polymerase activity. A single recessive nuclear mutation (mip1) is responsible for rho 0 phenotype and mtDNA polymerase deficiency in vitro. At 25 degrees C (or 30 degrees C) a dominant suppressor mutation (SUP) masks the deficiency in vivo. The meiotic segregants (mip1 sup) which do not harbor the suppressor have a rho 0 phenotype both at 25 and 35 degrees C. They have no mtDNA polymerase activity, in contrast with MIP rho 0 mutants of mitochondrial inheritance which do exhibit mtDNA polymerase activity. In the thermosensitive mutant (mip1 SUP), the replication of mtDNA observed in vivo at 30 degrees C is completely abolished at 35 degrees C. In the meiotic segregants (mip1 sup), no mtDNA replication takes place at 30 and 35 degrees C. The synthesis of nuclear DNA is not affected. DNA polymerases may have replicative and/or repair activity. There is no evidence that mip mutants are deficient in mtDNA repair. In contrast the MIP gene product is strictly required for the replication of mtDNA and for the expression of the mtDNA polymerase activity. This enzyme might be the replicase of mtDNA.     

Mitochondrial single-stranded DNA-binding proteins: in search for new functions

During the evolution of the eukaryotic cell, genes encoding proteins involved in the metabolism of mitochondrial DNA (mtDNA) have been transferred from the endosymbiont into the host genome. Mitochondrial single-stranded DNA-binding (mtSSB) proteins serve as an excellent argument supporting this aspect of the endosymbiotic theory. The crystal structure of the human mtSSB, together with an abundance of biochemical and genetic data, revealed several exciting features of mtSSB proteins and enabled a detailed comparison with their prokaryotic counterparts. Moreover, identification of a novel member of the mtSSB family, mitochondrial telomere-binding protein of the yeast Candida parapsilosis, has raised interesting questions regarding mtDNA metabolism and evolution.