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. 2023 May 8;21(1):103.
doi: 10.1186/s12915-023-01606-1.

Secondary structure of the human mitochondrial genome affects formation of deletions

Victor Shamanskiy #  1 , Alina A Mikhailova #  1 , Evgenii O Tretiakov #  2 , Kristina Ushakova #  1 , Alina G Mikhailova #  1   3 , Sergei Oreshkov  1 , Dmitry A Knorre  4 , Natalia Ree  1 , Jonathan B Overdevest  5 , Samuel W Lukowski  6 , Irina Gostimskaya  7   8 , Valerian Yurov  1 , Chia-Wei Liou  9 , Tsu-Kung Lin  9 , Wolfram S Kunz  10   11 , Alexandre Reymond  12   13 , Ilya Mazunin  14 , Georgii A Bazykin  14   15 , Jacques Fellay  16 , Masashi Tanaka  17   18   19 , Konstantin Khrapko  20 , Konstantin Gunbin  1   21 , Konstantin Popadin  22   23   24
Affiliations

Secondary structure of the human mitochondrial genome affects formation of deletions

Victor Shamanskiy et al. BMC Biol. .

Abstract

Background: Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions.

Results: By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9 kb and another within 13-16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp (base pair) and a second arm at 13,447-13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging.

Conclusions: Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

Keywords: Aging; Contact zone; Deletions; Direct repeats; Global secondary structure; Inverted repeats; Mitochondrial DNA; Single-stranded DNA; mtDNA replication.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Potential secondary structures formed by a single-stranded parental heavy chain during mtDNA replication. The lower panel shows that direct repeats, marked by black arrows, have different chances of being realized into deletions as a function of a spatial structure. The close spatial proximity of repeats (bold dotted lines) increases the probability of deletion formation, while for repeats that are spatially separated by a greater distance, this probability is decreased (thin dotted line)
Fig. 2
Fig. 2
Secondary structure of mtDNA. A Clusters of deletions within the major arc. The majority of clusters are located close to each other within the potential contact zone. The colors on the scheme on top correspond to the clusters. The vertical density plots on the right part of the figure demonstrate the distribution of deletion centers: real (observed) and random (expected). B Realized (red) versus non-realized (gray) repeats tend to be enriched in the potential contact zone. Mosaic-plot of repeats (realized versus non-realized) within and outside the potential contact zone. C Bottom-left: heatmap of the microhomologies between 100 bp windows within the major arc. Microhomology alone poorly explains the distribution of the deletions (empty circles). Top right: heatmap of the data-driven contact zone, based on the AIC of the compared models. D In silico approach for major arc global folding prediction. Bottom left triangle: contact matrix derived from the in silico folding estimation of the major arc's whole single-stranded heavy chain; top right triangle: contact matrix derived from the in silico folding estimation of 100 bp windows of the single-stranded heavy chain of the major arc
Fig. 3
Fig. 3
Integral scheme of the origin of mtDNA deletions. The parental heavy strand is marked by a gray line, while the parental light strand is marked by a black line. Daughter strands are indicated by dotted lines. Direct nucleotide repeats are marked by red arrows, and inverted nucleotide repeats are marked by green arrows. The origin of replication of the heavy strand (OH) and the origin of replication of the light strand (OL) are labeled. A At the start of replication, the daughter heavy strand (dotted gray line) is replicated on the template of the parental light strand (black line), and the replication fork begins to move from OH towards OL. B During this time, the parental heavy strand remains single-stranded (ssDNA) for a significant amount of time, and different types of microhomology, including inverted repeats (green arrows), can fold the spatial structure. C Once the first replication fork reaches OL, the second one begins replicating the daughter’s light strand (dotted back line) on the template of the parental heavy strand. D The second replication fork stalls near the stem initiated by the inverted repeats. If the stalling time is sufficiently long and the spatial structure of ssDNA cannot be resolved, the new-synthesized daughter chain dissociates partially and re-aligns with the second arm of the direct repeat. If ssDNA forms a stem, it may rotate (as indicated by the black arrow), potentially bringing direct repeats even closer together. E This allows the replication fork to continue replication. F Replication of the daughter light strand is finished, and the ssDNA loop region disappears either in the second round of mtDNA replication (see Fig. 5f in [28]) or when mtDNA with deletions is repaired, and ssDNA is degraded
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