Journal: The Journal of Biological Chemistry

Article Title: Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication

doi: 10.1016/j.jbc.2022.102196

Figure Lengend Snippet: SJCRH30 POLG Y955C cells contain less mtDNA and fewer mtDNA nucleoids than POLG wildtype cells. A , POLG wildtype (WT) and Y955C cells were separately dual stained with MitoTracker Red and PicoGreen dsDNA Reagent, and live-cell images were collected on a fluorescent microscope. Three P OLG WT and four Y955C PicoGreen -stained nuclei are labeled “N,” and three PicoGreen -stained mtDNA nucleoids are emphasized with white arrows in both cell types. For clarity, the PicoGreen ( green ) and MitoTracker Red ( red ) channels are shown separately in grayscale and together in color in the merged images. A representative image for each cell type is shown. Scale bars represent 10 μm. B , mitochondrial nucleoids from n = 64 total cells were counted for each cell line and from two different experiments (n = 32 cells for each) using different passages; ∗∗∗∗ p < 0.0001. C , BamHI-digested whole-cell extracted DNA samples were analyzed via Southern blot and nonradioactive probe hybridization. The blots were simultaneously probed with the DIG-labeled 18S nDNA probe (N, lower panels ; nucleotide positions 101–600) and the mtDNA-specific probe (MT, upper panel ; nucleotide positions 168–606). Bands were quantitated using the open-source image-processing package Fiji, as described ( , ). A representative blot is shown. On each blot, the average normalized band intensity values of WT mtDNA relative to nDNA on day 7 were set to 100%, and all other samples were compared with it. Statistical significance was determined by a two-way ANOVA, n = 12 (quadruplicates from three experiments using different passages, a total of six blots were analyzed, two per experiment); ∗∗∗∗, ####, and $$$$ p < 0.0001; ∗∗∗ p < 0.001; ## and ∗∗ p < 0.01.

Article Snippet: For PicoGreen staining of cellular DNA, growth medium was removed from MatTek dishes, and 2.5 ml of medium plus 3 μl/ml of Quant-iT PicoGreen dsDNA Reagent was gently overlayed.

Techniques: Staining, Microscopy, Labeling, Southern Blot, Hybridization

Journal: The Journal of Biological Chemistry

Article Title: Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication

doi: 10.1016/j.jbc.2022.102196

Figure Lengend Snippet: The SJCRH30 POLG Y955C cell line harbors additional mtDNA topoisomers sensitive to S1 nuclease. WCE DNA samples were treated with RNase A and digested with BglII to fragment nDNA (but not mtDNA). Where indicated, samples were treated with S1 nuclease (S1). Alkaline denaturation (Denaturing) before Southern blotting is necessary for hybridization of dsDNA with a single-stranded (ss) probe but can be omitted to assess potential single-stranded DNA species. For nondenaturing analysis (Non-denaturing), the gels were excluded from the denaturing solution step, but the other steps were performed. The blots were probed using the DIG-labeled mtDNA-specific probe (nucleotide positions 168–606). The untreated denatured samples (without S1 nuclease) contain major mtDNA topoisomers such as catenanes (Cat.), relaxed circles (RC), linears, and supercoiled (S.C.) molecules. Catenanes include HMW mtDNA well species (Well sp), mid-range-MW (MMW) catenanes, and low-MW (LMW) catenanes. Y955C cells accumulate a large amount of a replication intermediate (Rep. intermed. or RI) that localizes between the LMW catenanes and RCs, is sensitive to S1 nuclease, and is barely detectable in the WT. Under denaturing conditions, a unique species was seen below the linear mtDNAs in Y955C untreated samples and is sensitive to S1 nuclease digestion (S1 sens, highlighted with an arrow on the blot). A BamHI-digested WT sample was run in parallel as a control for linear ∼16.6-kb mtDNA. Data below the blots are mean values ± SD. Mean and SD values were calculated from n = 6 data points from two sets of blots, four in total (two denatured and two nondenatured, all photographed on the same image and probed with the same batch/concentration of probe, a representative blot for each is shown). The three replicates for each treatment shown are separate WCE DNA preparations from different passages of cells. The denatured and nondenatured blots were separately analyzed, and topoisomer differences among WT untreated, WT S1 nuclease treated, Y955C untreated, and Y955C S1 nuclease treated were determined (the rows of the table below the blots were compared). Three or more data sets per row were compared using a one-way ANOVA or a Welch’s ANOVA, while data sets of two ( e.g. , nondenatures MMW catenanes) were analyzed using a t test. Identical lowercase letters within a row ((a) versus (a) or (b) versus (b)) indicate no significant difference in the mean level of topoisomers, while different letters represent statistically significant differences. p < 0.05 is considered significant, and all p -values were <0.031.

Article Snippet: For PicoGreen staining of cellular DNA, growth medium was removed from MatTek dishes, and 2.5 ml of medium plus 3 μl/ml of Quant-iT PicoGreen dsDNA Reagent was gently overlayed.

Techniques: Southern Blot, Hybridization, Labeling, Control, Concentration Assay