Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes.

The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication

A New Splice Variant of the Mouse SIRT3 Gene Encodes the Mitochondrial Precursor Protein

BACKGROUND: Mammals have seven NAD-dependent protein deacetylases. These proteins, called sirtuins, are homologous to yeast Sir2, and are emerging as important regulators of lifespan and intermediary metabolism. Three mammalian sirtuins, SIRT3-5 are mitochondrial. Sirtuins are highly conserved between species, yet mouse SIRT3 was reported to be markedly shorter than its human counterpart and to lack the N-terminal mitochondrial targeting signal present in the human protein. RESULTS: We have isolated a novel mouse SIRT3 splice variant. This cDNA contains two translation initiation codons upstream of the originally reported start site. We show, using immunofluorescence and protein expression analysis that these longer variants are expressed and efficiently targeted to mitochondria, and that the processed forms of these longer variants are identical in size to the endogenous mouse SIRT3. We also show that the previously described form of SIRT3 is not mitochondrial. CONCLUSIONS: Our observations point to a high level of conservation of SIRT3 as a mitochondrial protein in mice and human and indicate that several previous studies, which addressed mouse Sirt3 function, need to be re-evaluated

Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes

The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication

Human Dna2 is a nuclear and mitochondrial DNA maintenance protein

Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance

Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle

PATATIN AS BINDER IN MEAT SUBSTITUTES - EP 3 944 769 A1

The invention provides a method for suppressing off-flavour formation in meat substitutes which have been bound with native patatin. The method comprises a) providing a mixture comprising water, a denatured pro- tein, native patatin and a lipid, which lipid is defined as a substance comprising fatty acid tri-esters of glycerol; b) shaping the meat substitute; and c) cooling the meat substitute to a temperature of from -35 °C to 20 °C; wherein the fatty acids in said lipid comprise less than 2 % by mass of fatty acids having a chain length of C12 or less. The invention also provides a meat substitute, comprising water, native patatin, a denatured protein and a lipid, which lipid is defined as a substance comprising fatty acid tri-esters of glycerol, wherein the fatty acids in said lipid comprise less than 2 % by mass of fatty acids having a chain length of C12 or less, and wherein preferably, the fatty acids comprise less than 2 % by mass of fatty acids having a chain length of C14 or less. This meat substitute has the advantage of minor off-flavor formation in tim

Mitochondria directly influence fertilisation outcome in the pig

The mitochondrion is explicitly involved in cytoplasmic regulation and is the cell's major generator of ATP. Our aim was to determine whether mitochondria alone could influence fertilisation outcome. In vitro, oocyte competence can be assessed through the presence of glucose-6-phosphate dehydrogenase (G6PD) as indicated by the dye, brilliant cresyl blue (BCB). Using porcine in vitro fertilisation (IVF), we have assessed oocyte maturation, cytoplasmic volume, fertilisation outcome, mitochondrial number as determined by mtDNA copy number, and whether mitochondria are uniformly distributed between blastomeres of each embryo. After staining with BCB, we observed a significant difference in cytoplasmic volume between BCB positive (BCB+) and BCB negative (BCB-) oocytes. There was also a significant difference in mtDNA copy number between fertilised and unfertilised oocytes and unequal mitochondrial segregation between blastomeres during early cleavage stages. Furthermore, we have supplemented BCB- oocytes with mitochondria from maternal relatives and observed a significant difference in fertilisation outcomes following both IVF and intracytoplasmic sperm injection (ICSI) between supplemented, sham-injected and non-treated BCB- oocytes. We have therefore demonstrated a relationship between oocyte maturity, cytoplasmic volume, and fertilisation outcome and mitochondrial content. These data suggest that mitochondrial number is important for fertilisation outcome and embryonic development. Furthermore, a mitochondrial pre-fertilisation threshold may ensure that, as mitochondria are diluted out during post-fertilisation cleavage, there are sufficient copies of mtDNA per blastomere to allow transmission of mtDNA to each cell of the post-implantation embryo after the initiation of mtDNA replication during the early postimplantation stages

Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle