Volume 65, Issue 3, Pages 527-538.e6 (February 2017) Single-Molecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion  Aaron F. Phillips, Armêl R. Millet, Marco Tigano, Sonia M. Dubois, Hannah Crimmins, Loelia Babin, Marine Charpentier, Marion Piganeau, Erika Brunet, Agnel Sfeir  Molecular Cell  Volume 65, Issue 3, Pages 527-538.e6 (February 2017) DOI: 10.1016/j.molcel.2016.12.014 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 65, 527-538.e6DOI: (10.1016/j.molcel.2016.12.014) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 A Single-Molecule DNA Combing Approach to Visualize mtDNA Replication (A) Schematic of mito-SMARD. Cells were sequentially pulse-labeled with media containing CldU and IdU, followed by a chase period. mtDNA was purified and digested with the AleI restriction enzyme, which cleaves upstream of OriH. Linearized DNA was then stretched on a silanized glass slide and stained with antibodies that distinguish CldU (green) from IdU (red). (B) Left: stretched mtDNA molecules marked with a fluorescent DNA intercalator (YOYO-1). Center: examples of mtDNA molecules from HT1080 cells labeled with mito-SMARD and visualized with conventional microscopy (Nikon Eclipse Ti-V) using a 100×/1.45 NA oil objective. Shown are mtDNA molecules replicating in the presence of CldU (green molecules) and IdU (red molecules) as well as those replicating during the switch between CldU and IdU (dually labeled molecules). Right: mtDNA molecules detected by mito-SMARD and imaged with an OMX 3D-SIM super-resolution microscope (Applied Precision). Molecular Cell 2017 65, 527-538.e6DOI: (10.1016/j.molcel.2016.12.014) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Mito-SMARD Highlights the Mode of mtDNA Replication In Vivo (A) Predicted pattern of CldU and IdU incorporation based on the strand-coupled or strand displacement models. Taking into consideration dual-labeled molecules, the strand-coupled model is expected to yield one replication pattern in which green is followed by red. In contrast, according to the strand displacement model, two independent groups of molecules will be visualized: one labeled with green followed by red (group A) and an equivalent fraction with red flanked by green on either side (group B). (B) Mito-SMARD analysis of mtDNA in HT1080 cells yields molecules that are categorized in two distinct groups: group A molecules show CldU (green) followed by IdU (red) labeling, whereas group B molecules show an IdU-CldU-IdU labeling pattern. (C) Quantification of the frequency of group A versus group B molecules in PolrmtF/F and Polrmt+/+ cells with the indicated treatment. Cells were analyzed 48 hr after induction of a 4-hydroxytamoxifen-inducible Cre-ERT2. Average frequencies were calculated from three independent experiments (n ≥ 100 molecules in each experiment) with SD. Molecular Cell 2017 65, 527-538.e6DOI: (10.1016/j.molcel.2016.12.014) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Evidence for Replication Fork Stalling in Cells Expressing Mutant Twinkle (A) Genotyping PCR to detect heterozygous TwinkleR374Q/+ generated by CRISPR/Cas9 targeting of RPE-1. The PCR product was treated with a restriction enzyme that specifically cleaves the targeted allele. Two independently derived clonal cell lines were generated. Targeting was confirmed by Sanger sequencing of cDNA from the indicated clones. The asterisks mark mutated residues, including the R374Q mutation and a silent mutation in the protospacer adjacent motif (PAM) sequence. (B) Schematic for mito-SMARD to assess replication fork stalling. FISH probes that label the control region of the mitochondrial genome were used to mark the end of the linearized mtDNA molecules stretched on glass slides. Top: schematic of molecules representative of fully replicated mtDNA. Bottom: schematic of partially labeled molecules indicative of replication fork stalling. (C) Representative mtDNA molecules labeled with CldU (green), IdU (red), and CldU-IdU (green-red) and stained with mito-FISH probes (blue). (D) Evidence of replication fork stalling in the major mtDNA arc in TwinkleR374Q/+ and TwinkleY508C/Y508C cells. Shown are mtDNAs obtained from cells expressing mutant and wild-type Twinkle. Top: examples of fully replicating mtDNA molecules with IdU/CldU incorporation patterns consistent with replication initiation from OriL and OriH. Bottom: molecules represent labeling patterns consistent with fork stalling. It is important to note that the duration of the IdU pulse (90 min) allows mtDNA molecules that have initiated replication during the CldU pulse to fully complete synthesis (rate of mtDNA synthesis, ∼190 nt/min) (data not shown; Clayton, 1982; Korhonen et al., 2004). Molecular Cell 2017 65, 527-538.e6DOI: (10.1016/j.molcel.2016.12.014) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Engineering TALENs to Induce the Mitochondrial Common Deletion (A) Schematic of mtDNA with cleavage sites for each mito-TALEN (D-loop, CD3′, CDmid, CDin, CD5′, CDout). Indicated in red are the two 13-bp repeats surrounding the common deletion (CD). The primers used for PCR amplification of the common deletion are also shown. (B) Immunofluorescence microscopy of U2OS cells transfected with mito-TALENs that target CD5′ (left) and CD3′ (right). Hemagglutinin (HA)-tagged mito-TALEN was visualized 48 hr after transfection (top), MitoTracker marks mitochondria (center), and DAPI marks nuclei. (C) Fractionation experiments followed by western blot analysis to detect mito-TALENs (HA tag) in the cytoplasm (C) and mitochondria (M). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) marks the cytoplasmic fraction, and voltage-dependent anion-selective channel (VDAC) denotes the mitochondrial fraction. (–ve), no mito-TALEN. (D) PCR detection of mtDNA4977 in U2OS cells treated with the indicated mito-TALEN and harvested 7 days after transfection. The asterisk marks a catalytically inactive mito-TALEN-CD5′. (E) PCR detection of the common deletion in cells treated with the indicated mito-TALEN nickase (Nick-H induces a break in the heavy strand, Nick-L induces a break in the light strand) and nuclease (DSB). Molecular Cell 2017 65, 527-538.e6DOI: (10.1016/j.molcel.2016.12.014) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Replication-Mediated Repair Leads to the Common Deletion (A) qPCR analysis of mtDNA4977 in cells treated with mito-TALEN-CD5′. The amount of mtDNA4977 was determined 7 days after mito-TALEN transfection and normalized to total mtDNA (12S). The frequency of mtDNA4977 in cells treated with siRAD51 is presented relative to siControl. (B) Relative percentage of mtDNA4977 accumulation in LIG1F/− and LIG3F/− cells after Cre induction and mito-TALEN expression. (C) qPCR analysis of mtDNA4977 induced by mito-TALEN-CD5′ in cells depleted for the indicated nucleases and analyzed 7 days after transfection. (D) Induction of mtDNA4977 upon inhibition of replication in cells treated with the indicated siRNA and analyzed 7 days after transfection. The frequency of mtDNA4977 in all genetic perturbations was normalized to mtDNA content (12S) and is presented relative to siControl. Values represent the mean of three independent experiments with SD. Molecular Cell 2017 65, 527-538.e6DOI: (10.1016/j.molcel.2016.12.014) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 The Evolution of Repeats in the Mitochondrial Genome (A) Phylogenetic distribution of mtDNA repeats of 142 species. Phylogenetic relationships were analyzed through the NCBI Taxonomy Database, and organisms are divided into evolutionary groups. Bars represent the total number of direct and inverse repeats (>12 bp) normalized to the size of the genome. (B) Bar graph representing the average number of repeats compared with a theoretical number computed for each evolutionary group, taking into account the average size of the mitochondrial genome and the percentage of GC content. Molecular Cell 2017 65, 527-538.e6DOI: (10.1016/j.molcel.2016.12.014) Copyright © 2017 Elsevier Inc. Terms and Conditions