Comparative analysis of mtDNA from various human tissues by 1D‐ and 2D‐AGE (IV,

For comparative analysis, 2DNAGE of the mtDNA replication intermediates from various human tissues was performed. In order to get a whole mitochondrial genome view, a single-cleavage restriction enzyme approach was applied using gel conditions capable of resolving 16.6 kb fragments. I was able to detect intact mitochondrial theta–like replication intermediates in total DNA preparations of post mortem brain, skeletal muscle (iliopsoas) and kidney that were comparable in quality to the ones from freshly prepared total DNA sample from HEK293T cells (Figure 5.7). In the BamHI digest typical “eyebrow”-forms, indicating RITOLS replication, were also detected (see Supplementary Figure 1 of IV). In the autopsy samples from some tissues, such as liver and lymph node, the mtDNA was generally too degraded to detect any replication intermediates (data not shown).

In heart there were no replication intermediates corresponding to the expected products of theta-replication on single-cleavage restriction enzyme gels, the major replication intermediates seen migrated on a Y-like arc, encompassing the whole mtDNA molecule (Figure 5.7A:i, 5.7B:i).

The products of PvuII and BamHI digests were indistinguishable. The majority of other non-linear molecules appeared to be recombination intermediates migrating on a standard X-arc, as already reported in Kajander et al. (2001) for smaller fragments of the genome. Some complex X-like forms of more than twice the genome size and probably corresponding to three or more recombining molecules were also detected (denoted as dx in Figure 5.7). Similar X forms as well as a regular X-arc were also present together with theta-forms in brain samples. Identical results were obtained with sucrose gradient-purified mtDNA from human cardiac muscle and cortex obtained as soon as possible after death (6h), which were later used in TEM (for details see Supplementary Figure 3 in IV).

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Figure 5.7. 2DNAGE of total DNA shows theta-type mitochondrial replication intermediates (RIs) in human brain, skeletal muscle, kidney and and cultured cells, but not in heart. See chapter 4.20.2 for interpretation. (A) PvuII (nt 2564) digested DNA from various tissues and HEK293T cells. All other panels except heart (A-i) show theta-type replication intermediates. (B) Identical samples digested with BamHI (nt 14259). Note the similarity of (A-i) and (B-i). In heart most of the non- linear forms migrate on X- and double-Y-arcs. Some more than 2n genome length Xs are present (dx). In BamHI digests (bottom row) the dominant RIs are double-Ys (dy) and tailed circles (t) as depicted in Figure 4.6B and 4.6C. Again, the heart RIs migrate as a Y-like arc. Brain mtDNA 2DNAGE patterns show both theta- and heart-like replication intermediates. Note the low amount of any replicative forms detected in skeletal muscle mtDNA.

2DNAGE of smaller fragments revealed further tissue differences in replication intermediates (Figure 2 in IV). As expected, 2DNAGE of purified mtDNA from cultured cells (Figure 2b:vi in IV) shows typical slow-moving-Y (SMY)-like arcs indicative of RITOLS intermediates. These intermediates were preserved in total DNA preparations from the same cells (Figure 2b:v in IV) together with some partially degraded RNA-rich intermediates (mSMY). Traces of these same intermediates could also be seen in total DNA samples obtained from autopsied kidney (Figure 2b:iv in IV). Heart and brain samples however did not show any recognizable RITOLS intermediates, instead the Y-arc was well defined and X-forms were abundant, as already seen in the PvuII and BamHI digests. The high MW X-like structures (dx) were also present in these preparations. Furthermore, no initiation-arcs were detected from OH-containing or any other fragments of human heart mtDNA.

The X-forms were seen in all 12 healthy human individuals checked so far and seemed to be age-independent although no precise quantifications were done (Supplementary Figure 3 of IV). Interestingly, the abundant X-forms in heart seem to be a human-specific feature, since no

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X-forms were detected from cardiac muscle total DNA samples from mouse, rabbit or pig (Supplementary Figure 3 in IV).

On one-dimensional Southern blots of undigested human heart mtDNA, most material migrated in two main positions that are denoted as bands s (for slow) and f (for fast) (Figure 5.8A).

The fastest migrating band represents one genome length, topoisomerase I -sensitive supercoils (see also Figure 5.12). The other easily recognizable form is the one genome length (16.6. kb) linear.

A better separation was achieved by applying similar 2DNAGE conditions as for the single-cleavage analysis (Figure 5.8B). This method revealed that band f is actually a heterogeneous mixture of different molecular forms, including 33 kb linears (2n), one genome-length open circles (oc) and topoisomers of larger, most probably two genome length circles. As seen in Figure 5.8B-:i the one genome-length supercoils (sc) migrated more slowly in the second dimension, with the same mobility as open circles. Similarly the bulk of band f migrated more slowly in the second dimension, and ran in the same position as the bulk of band s. When topoisomerase I or IV was applied most of the band f disappeared together with the supercoils (Figure 5.8A, B:ii). It should be noted that besides decatenation, topoisomerase IV also relaxes supercoiled molecules.

Figure 5.8. Human cardiac muscle mtDNA is organized in various topological forms consisting of 16.6 and 33 kb molecules. (A) A Southern blot of total DNA from human heart, probed with mitochondrial probes, showing different enzymatic treatments. u – untreated, ti – topoisomerase I, tiv – topoisomerase IV (a decatenase), t7 – T7 endonuclease I, exo – lambda phage exonuclease III.

Most of the material in the untreated sample is concentrated in diffuse bands denoted t and f. Band f has a linear mobility of around 30-35 kb, while t migrates slower than a 48.5 kb lambda phage genome fragment. (B) Enzymatic digests as well as 2DNAGE reveal that band f actually consists of multiple forms, which include 33 kb linear molecules, one genome length circles, junctional molecules and topoisomers. (i) Untreated, (ii) topoisomerase IV, (iii) topoisomerase IV and T7 endonuclease I, (iv) T7 endonuclease I. 2n - two genome length, 1n - one genome length, h – heterogeneous large molecules, oc – open circles, sc – supercoils, x - junctional molecules.

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Topoisomerase IV treatment revealed an additional species, x, migrating around 33 kb in the first dimension. This species quite likely represents a circle recombinined with a linear molecule, since circular molecules would be slower in the first dimension and fully linear partners would form an X-arc. Circular recombining molecules as well as 33 kb circles would likely migrate in the same position as catenated 16.6 kb circles (see Figure 5.12.). In the gels presented here, their presence is masked by band s, which most likely contains all these forms. This notion is supported by the fact that T7 endonuclease I treatment resulted in an increase in 16.6 kb linears and when applied together with topoisomerase IV left a defined residual band (Figure 5.8B:iii-iv). This band has the migration properties of a circle and is resistant to decatenation by topoisomerase IV as well as cruciform cutting endonuclease, thus it is safe to assume that it represents 33 kb circles (2n oc).

Topoisomerase IV and T7 endonuclease resolved the heterogeneous material above band s (denoted as h) producing 33 and 16.6 kb linears, indicating that this material includes catenanes and recombining molecules of one and two genome lengths. Phosphoimager analysis of these residual circles and linears showed that around two-thirds of the total signal corresponds to 33 kb sized molecules. This is in agreement with the EM data, because dimeric genomes logically give twice the signal.

Mitochondrial DNA from other tissues, including brain, differed from heart in appearance on Southern blots of one-dimensional gels of uncut DNA (Figure 5.9). Most of the material in all of these tissues consisted of 16.6 kb open circles. Some low mobility bands possibly corresponding to band s in heart were also seen in samples from these tissues, but were mainly topoisomerase IV-sensitive, two molecule catenanes as seen in cultured cells. High molecular weight forms were generally absent and the amount of supercoiled genomes was relatively high in the skeletal muscle sample. As mentioned before, DNA from autopsy-derived lymph node and liver samples was most degraded (data not shown).

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Figure 5.9. The organization of mtDNA differs between various human tissues. (a) A Southern blot of total DNA from various human tissues probed with a mtDNA specific probe. (i) Heart, (ii) brain, (iii) skeletal muscle and (iv) kidney. In other tissues band f is clearly defined and corresponds to open circles of one genome size and band t represents a pair of one genome-sized relaxed catenated circles. Skeletal muscle has relatively high amount of supercoiled one genome size molecules.

5.2 Effects of mtDNA-binding proteins on mtDNA replication and