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Review
. 2024 Jun 5;481(11):683-715.
doi: 10.1042/BCJ20230262.

Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation

Tiago M Bernardino Gomes  1   2   3 , Amy E Vincent  1   2 , Katja E Menger  2   4 , James B Stewart  2   4 , Thomas J Nicholls  2   4
Affiliations
Review

Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation

Tiago M Bernardino Gomes et al. Biochem J. .

Abstract

Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.

Keywords: DNA damage; DNA replication and recombination; mitochondrial dysfunction; mtDNA.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1.
Figure 1.. Human mtDNA structure.
The positions of mtDNA-encoded genes are shown separated according to whether they are encoded by the heavy strand (H-strand) or light strand (L-strand), and the location of the replication origins OriH and OriL are indicated. An enlargement of the non-coding region (NCR, top) shows the location of the three promoters HSP, LSP and LSP2, as well conserved sequence blocks (CSBs) 1, 2 and 3, and 7S DNA, with its 5′ end in the OriH region and its 3′ end at the termination-associated sequence (TAS).
Figure 2.
Figure 2.. Mechanisms of mtDNA replication.
(A) Replication priming in the OriH region. Transcription from LSP by POLRMT creates a hybrid R-loop anchored at CSB2, which is processed by RNASEH1 to generate a 3′ RNA end that can be utilised by POLγ for DNA synthesis. (B) Replication priming at OriL. When exposed in ssDNA form, the OriL sequence forms a stem-loop structure. POLRMT initiates RNA synthesis from a poly(T) stretch in the loop of this structure, generating a primer for DNA synthesis by POLγ. (C–E) Models of mtDNA replication. (C) The strand displacement model (SDM). Leading (heavy) strand replication is initiated at OriH, and the displaced lagging strand template is coated with MTSSB (yellow). Lagging strand replication is initiated by POLRMT, which forms a primer at a stem-loop structure at the OriL site, allowing continuous L-strand synthesis. (D) Strand coupled replication. Initiation is associated with a broad zone downstream of the NCR, Ori-z. Replication of the H-strand is continuous, while L-strand replication requires the formation of discontinuous Okazaki fragments. (E) Ribonucleotide incorporation throughout the lagging strand (RITOLS). Replication is initiated in the NCR, with OriL being the major site of lagging-strand DNA synthesis. The displaced lagging-strand template is coated with RNA transcripts (bootlaces) rather than MTSSB. (F) Hemicatenated mtDNA replication products are separated by TOP3A.
Figure 3.
Figure 3.. Rearrangement breakpoint patterns in mammalian mtDNA.
All reported human mtDNA breakpoints in the MitoBreak database [168] were plotted using Circos (A), or categorised by association with single, large-scale deletion disorders (B), multiple mtDNA deletion disorders (C), ageing (D), or Parkinson's disease (E). All reported breakpoints in mouse mtDNA (F) are shown for comparison. The outer track indicates the locations of mitochondrial mRNAs (white), tRNAs (black), rRNAs (dark grey) and the NCR (light grey).
Figure 4.
Figure 4.. Mechanisms of mtDNA deletion formation.
(A) Representation of mtDNA, containing direct repeat (DR) sequences (blue and orange boxes) in the major arc. (B) Slip-replication model. Mispairing of an upstream repeat sequence in the displaced lagging-strand template, followed by breakage of the displaced loop (red arrows), generates a truncated template molecule that is replicated to generate a deletion-containing mtDNA. (C) Copy-choice recombination model. Slippage and mispairing during lagging-strand mtDNA replication forms a heteroduplex molecule containing a single-stranded loop. Replication of this molecule generates one full-length mtDNA molecule and one deletion-containing molecule. (D–F) Models of mtDNA deletion formation associated with strand breaks. Generation of a double-strand break (DSB) in mtDNA (D), followed by partial degradation of the broken DNA ends, creates truncated mtDNA molecules. Pathways are shown both for homology-mediated annealing of resected DNA ends (E) and the ligation of partially-degraded DNA ends without homology (F).
Figure 5.
Figure 5.. Clinical manifestations of mtDNA instability.
Symptoms associated with either single, large-scale mtDNA deletion disorders or mtDNA maintenance disorders are shown that affect the central nervous system [14,231,233–237,309–311], eyes and ears [14,231–238,243,312], heart [14,179,231,233–235,237,287,290–292], gastrointestinal tract [14,231,233–235,237,313], endocrine system [14,231,233–236,314,315], kidney [14,231,233–235,316,317], neuromuscular system [14,231–238,243], as well as other complications [14,231,233–237].
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