DNA

3.2.1 Total DNA extraction (I, II and III)

For total DNA extraction, cells were washed once with PBS, harvested in 1 mL of PBS and then pelleted by centrifugation for 3 min at 16 000 x g at 4ºC. Cells were lysed overnight at 37ºC with 400-1000 µL of lysis buffer containing 10 mM Tris pH 7.4, 10 mM EDTA, 150mM NaCl, 0,4% SDS and 0,5 µg/µL Proteinase K.

On the next day, protein traces and lipids were separated from the DNA by two phenol:chloroform extractions with one volume of Phenol:Chloroform:isoamyl alcohol (25:24:1) (Biotechnology grade, Amresco) followed by a single step using chloroform in order to remove traces of phenol. Next, the DNA was precipitated with two volumes of cold 100% ethanol and 0,1 volume of 3 M sodium acetate and incubated for one hour at - 20ºC. The precipitated DNA was pelleted by centrifugation for 20 min at 16 000 x g at 4ºC and washed once with 70% ethanol to remove salts. The DNA pellet was resuspended overnight at 37ºC in 100-200 µL 1x FastDigest buffer (ThermoFisher Scientitic) and 1 µL of FastDigest BglII

73 (for human DNA) or FastDigest KpnI (for mouse DNA) which cut nuclear DNA but not mtDNA. Next day, the DNA concentration was determined with a NanoDrop^TM1000 spectrophotometer.

3.2.2 mtDNA copy number determination by Taqman-based qPCR (II, III)

Total DNA was isolated as described before. In order to increase the efficiency of the PCR reaction and prevent variations caused by different DNA topologies the DNA samples were fragmented by sonication using a ultrasonic processor UP200S (Hielscher) set at 0,5 cycles and 90% amplitude. 100 ng of the sonicated total DNA were measured by triplicate using 0,1 µM mitochondrial and 0,5 µM nuclear primers pre-mixed with 125 nM fluorescent FAM mtDNA probe and HEX nDNA probe in 20 µl AccuStartII PCR supermix (Quanta) and the following thermoprofile: 5 min at 95°C followed by 40 cycles of a 3-step amplification:

denaturation at 95°C for 20 s, annealing at 58°C for 20 s and elongation at 72°C for 20 s. The signal from the probes was detected using the AriaMx Real-Time system (Agilent Technologies) and the data was analyzed with the associated software Agilent AriaMx 1.0. mtDNA quantification values were corrected with nDNA values. The primers used were:

3.2.3 mtDNA copy number determination by Southern blotting (I, II)

2 µg of total DNA digested for at least 6 hours at 37ºC with FastDigest BamHI (ThermoFisher Scientitic) for human DNA or FastDigest HindIII for mouse DNA (which respectively cut mtDNA only once) were separated over a 0.4% agarose

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gel in 1x TBE buffer at 20-30 V for at least 16 hours at room temperature. Southern blotting was performed as follow: the gel was washed twice with depuration buffer (0,25 M HCl) for 15 min to break down the glycosidic bonds, followed by two washes with denaturation buffer (0,5 M NaOH and 1,5 M NaCl) for 25 min to separate the DNA strands and facilitate the transfer. The DNA was then transferred overnight by gravity onto a nylon membrane (Amersham Hybond-XL). Next day, the membrane was neutralized with 6x SSC and the DNA cross-linked onto the membrane by baking for 2 hours at 80ºC.

Membranes were probed with ND5 (nts 12 992–13 670) for human mtDNA or Cytb (nts 14 783–15 333) for mouse mtDNA and 18S rDNA (nts 850-1347 of human 18S, accession NR_0032862, or nts 166-613 of mouse 18S, NR_003278) as loading control. DNA probes were initially generated by PCR spanning the indicated nucleotides (Table 1) and then labelled with a random primed labelling system (Rediprime^TMII, Amerhsam^TM, GE healthcare) and (α-³²P) dCTP (3000 Ci/mmol).

After pre-hybridizing the blots with hybridization buffer (250mM NaPⁱ pH 7.2, 7% SDS, 2 mM EDTA) for 15 min at 65ºC and rotation, the labelled, denatured probe was added and blots were hybridized overnight in the same conditions.

On the next day, the non-specifically bound probe was washed off by four washes with 1x SSC and 0,1% SDS for 20 min at 65ºC in rotation. Finally, the radioactive blot was exposed to a KODAK storage phosphor screen SO230, and the radioactive signal was detected using the Molecular Imager FX (BioRad) and analyzed using the associated QuantityOne software.

Table 1. List of probes, coordinates and PCR primer sequences used. ND5, ND3, ND2, NCR, 7S and Cytb are mtDNA encoded genes or regions, while 18s is the genomic DNA gene used as loading control for mtDNA copy number quantification.

Name Source Coordinates Sequence

ND5 Human 12,992 Fw 5´- CAGCAGCAGGCAAATCAG 13,670 Rv 5´-TCGTTAATGTTAGTAAGGGTGG ND3 Human 10,131 Fw 5´ -ACCACAACTCAACGGCTACA

10,382 Rv 5´- TGTAGTCACTCATAGGCCAGAC ND2 Human 4,470 Fw 5´-GTTATACCCTTCCCGTACTA

5,511 Rv 5´-TATTTAACCTAAATTTCTAT

NCR Human 37 Fw 5´-AGCTCTCCATGCATTTGG

611 Rv 5´-CAGTGTATTGCTTTGAGG 7S Human 16177 Fw 5´- ATCAAAACCCCCTCCCCAT

40 Rv 5´- AGCTCCCGTGAGTGGTTA 18s

(nDNA) Human 850 Fw 5´- CCGCAGCTAGGAATAATGGA 1347 Rv 5´- AACTAAGAACGGCCATGCAC Cytb Mouse 14,783 Fw 5´-CCCAACAGGATTAAACTCAG

15,333 Rv 5´-CTTGAGAAGAGAAGATCTT

75 3.2.4 mtDNA species quantification by Southern blotting and restriction mapping (III)

To analyze the different mtDNA species (intact mtDNA, 11kb linear fragment, duplications and 7s DNA), 2 g of total DNA were digested at 37ºC for at least 6 hours with FastDigest HindIII and AflI (ThermoFisher) and then separated over a 0,8% agarose gel in 1x TAE buffer at 25 V for at least 16 hours at room temperature. To separate the 7s DNA fragment and facilitate its visualization the samples were heated for 10 min at 65ºC before being loaded into the gel. Southern blot was performed as before and the blot was probed with 7s (nts 12 992–13670).

The radioactive signal was captured as described before.

3.2.5 mtDNA topology analysis by Southern blotting (I, III)

To analyze mtDNA topology, 2 µg of uncut total DNA were separated over a 0,4% agarose gel as described before. Southern blotting was performed as previously and the membrane was probed with ND5 (nts 12 992–13 670). The radioactive signal was captured as described before.

3.2.6 Long-range DNA damage Taqman-based qPCR (II)

Total DNA was isolated as described previously but the mtDNA restriction step was skipped. 100 ng of total DNA were measured by quadruplicates using AccuStartII PCR supermix (Quanta) and 0,5 µM primers pre-mixed with 125 nM fluorescent FAM mtDNA probe and HEX nDNA probe. Two independent PCRs were run with the same DNA samples, amplifying a short (108 bp) and a long fragment (6,5 kb). Damage on the template DNA should reduce the performance of the long PCR, but barely affect the short PCR as the probability of the short fragment to contain a DNA lesion is rather small. The following thermoprofiles were used: Short PCR: 3 min at 95 ºC followed by 40 cycles of a 2-step amplification at 95 ºC for 10 s and 60 ºC for 20 s; Long PCR: 5 min at 94 ºC followed by 40 amplification cycles of denaturating at 94 ºC for 30 s, annealing at 64 ºC for 30 s and elongation at 70 ºC for 7 min. The signal from the fluorescent probes was detected using the AriaMx Real-Time system (Agilent Technologies) and the data was analyzed with the associated software Agilent AriaMx 1.0. The nuclear quantification values were used to correct the loading of the samples and the long-amplicon value was normalized against the short.The primers used were:

NDUFV1 nuclear gene, accession number U3BG69 Fw 5’ ATC CAG GAT CCC ACA GAG CT Rv 5’ CCT TTC CAG CAG ATG TGG GT

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Probe: 5’ VIC- GAG CCT TAG GGA AGA GGC AG –MGBNFQ 6,5 kb mitochondrial fragment

1978 Fw: 5’ – TGC CTG CCC AGT GAC TAA AG 8496 Rv: 5’ – GGT AGC TGT TGG TGG GCT AA

Probe 2017 Rv: 5’ Fam– TGA CCG TGC AAA GGT AGC AT-BHQ1 108 bp mitochondria fragment

2086 Rv: 5’ – GAC CCT CGT TTA GCC GTT CA 1978 FW: 5’ – TGC CTG CCC AGT GAC TAA AG

Probe 2017 Rv: 5’ Fam– TGA CCG TGC AAA GGT AGC AT-BHQ1