Discovery of a Major D-Loop Replication Origin Reveals Two Modes of Human mtDNA Synthesis

Mammalian mitochondrial DNA (mtDNA) replication has long been considered to occur by asymmetric synthesis of the two strands, starting at the multiple origins of the strand-displacement loop (D-loop). We report the discovery of a major replication origin at position 57 in the D-loop of several human cell lines (HeLa, A549, and 143B.TK^–) and immortalized lymphocytes. The nascent chains starting at this origin, in contrast to those initiated at the previously described origins, do not terminate prematurely at the 3′ end of the D-loop but proceed well beyond this control point, behaving as “true” replicating strands. This origin is mainly responsible for mtDNA maintenance under steady-state conditions, whereas mtDNA synthesis from the formerly identified D-loop origins may be more important for recovery after mtDNA depletion and for accelerating mtDNA replication in response to physiological demands

Decreased Reactive Oxygen Species Production in Cells with Mitochondrial Haplogroups Associated with Longevity

Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function. Zhang and colleagues found a strikingly higher frequency of a C150T transition in the D-loop of mtDNA from centenarians and twins of an Italian population, and also demonstrated that this base substitution causes a remodeling of the mtDNA 151 replication origin in human leukocytes and fibroblasts [1]. The C150T transition is a polymorphism associated with several haplogroups. To determine whether haplogroups that carry the C150T transition display any phenotype that may be advantageous for longevity, we analyzed cybrids carrying or not the C150T transition. These cybrids were obtained by fusing cytoplasts derived from human fibroblasts with human mtDNA-less cells (ρ^0 cells). We chose for cybrid construction and analysis haplogroup-matched pairs of fibroblast strains containing or not the C150T transition. In particular, we used, as one pair of mtDNA donors, a fibroblast strain of the U3a haplogroup, carrying the C150T transition and a strain of the U-K2 haplogroup, without the C150T transition, and as another pair, fibroblasts of the J2b haplogroup, carrying the C150T transition and of the J1c haplogroup, without the C150T transition. We have found no association of respiratory capacity, mtDNA level, mitochondrial gene expression level, or growth rate with the presence of the C150T transition. However, we have found that the cybrids with haplogroups that include the C150T transition have in common a lower reactive oxygen species (ROS) production rate than the haplogroup-matched cybrids without that transition. Thus, the lower ROS production rate may be a factor in the increased longevity associated with the U and the J2 haplogroups. Of further interest, we found that cybrids with the U3a haplogroup exhibited a higher respiration rate than the other cybrids examined

Mitochondrial DNA involvement in human longevity

AbstractThe main message of this review can be summarized as follows: aging and longevity, as complex traits having a significant genetic component, likely depend on a number of nuclear gene variants interacting with mtDNA variability both inherited and somatic. We reviewed the data available in the literature with particular attention to human longevity, and argued that what we hypothesize for aging and longevity could have a more general relevance and be extended to other age-related complex traits such as Alzheimer's and Parkinson's diseases. The genetics which emerges for complex traits, including aging and longevity, is thus even more complicated than previously thought, as epistatic interactions between nuclear gene polymorphisms and mtDNA variability (both somatic and inherited) as well as between mtDNA somatic mutations (tissue specific) and mtDNA inherited variants (haplogroups and sub-haplogroups) must be considered as additional players capable of explaining a part of the aging and longevity phenotype. To test this hypothesis is one of the main challenge in the genetics of aging and longevity in the next future

Analysis of O₂ consumption in digitonin-permeabilized cells of the cybrid clones using different substrates and inhibitors.

<p>The activities of the various components of the respiratory chain were investigated by measuring the respiration rate dependent on (<b>A</b>) malate plus glutamate, on (<b>B</b>) succinate plus G3P (in the presence of rotenone) and on (<b>C</b>) TMPD plus ascorbate (in the presence of antimycin). Cell lines are grouped by haplogroup. A total of 3–4 determinations were made on each of the three or four cybrid clones derived from each fibroblast strain. The mean of those determinations is shown. The error bars indicate the standard error of the mean. Horizontal gray lines represent the average for each haplogroup. Horizontal black bars with asterisks indicate differences between averages are statistically significant; *, P≤0.05; **, P<0.01. P = 0.0578 for the difference between the means of respiration rate of the J2b and the J1c cybrids in panel <b>A</b>, i.e. not quite statistically significant.</p

Mitochondrial protein synthesis rates.

<p><b>A</b>, Electrophoretic patterns of the mitochondrial translation products of cybrid clones labeled for 45 min with [³⁵S]methionine in the presence of 100 µg/ml of emetine. Each lane represents a different cybrid clone, the name of which is given below the corresponding bar in panel <b>B</b>. ND1, -2, -3, -4, -4L, -5, and -6, NADH dehydrogenase subunits 1, 2, 3, 4, 4L, 5, and 6, respectively; CYTb, apocytochrome <i>b</i>; COI, -II, and -III, subunits I, II, and III, respectively of cytochrome <i>c</i> oxidase; A6 and A8, subunits 6 and 8, respectively, of the H⁺-ATPase. <b>B</b>, Quantification of the labeling of the mitochondrial translation products shown in panel <b>A</b>. The cell lines are grouped by haplogroup, namely U3a, U-K2, J2b, and J1c. The horizontal gray lines in panel <b>B</b> indicate the mean level of labeling for each group of cybrids. Neither the difference between the U3a and the U-K2 cybrids nor the difference between the J2b and the J1c cybrids is statistically significant.</p

ROS generation rate.

<p>Cells of each cybrid clone were stained with the ROS-activated dye CM-H₂DCFDA. Fluorescence data were collected on a Molecular Devices fluorescence plate reader for 1 h. A total of 2–5 rate determinations were made on each cybrid clone. Each bar represents the average fluorescence production rate for the indicated cybrid clone. The error bars indicate the standard error of the mean. The haplogroup is indicated below each group of cybrid clones. Horizontal gray lines represent the average for each haplogroup. Horizontal black bars with asterisks indicate differences between averages are statistically significant, as determined by a two-tailed unpaired t-test; *, P≤0.05; **, P<0.01.</p

Membrane potential.

<p>Cybrid cell lines were stained with the fluorescent membrane potential indicator dye TMRE. The fluorescence was quantified by flow cytometry. A total of 2–3 determinations were made on each cybrid cell line and the mean values are shown. The error bars indicate the standard error of the mean. Gray bars represent the average for each haplogroup. Cell lines are grouped by haplogroup. No difference between any two groups of cybrids is statistically significant.</p

Control Region Polymorphisms in Cybrid Cell Lines.

<p>The table lists all the Control Region polymorphisms found in the sequences of the mtDNAs of cybrids TF3A5, E8, T8, F8, and TFA7. The haplogroup and subhaplogroup with which the polymorphism is associated is indicated. Most of these Control Region polymorphisms are haplogroup-specific.</p

Growth rates of cybrid cell lines.

<p>The population doubling times during 7 days of growth are indicated. The cell lines are grouped by mtDNA donor cell and the haplogroup of the donor is indicated, namely U3a, U-K2, J2b, and J1c. The horizontal gray lines indicate the mean doubling time for each group of cybrids. A black horizontal line indicates that the difference between the doubling times of the cybrid groups at the ends of the line is significant. ** indicates P≤0.01, by the t-test.</p