RESEARCH

Mitochondria – the energy factories of our cells – contain their own distinct genome that encodes for key components of the machinery that carries out oxidative phosphorylation. This genome, mtDNA, is only ca 16 kbp long in humans but is essential for efficient energy production. MtDNA defects such as mutations, deletions or depletion cause a wide variety of rare genetic disorders, and have also been associated with more common disease states like neurodegenerative diseases and some cancers.

MtDNA instability

The mtDNA is constantly exposed to various endogenous and exogenous insults that jeopardize its stability and may lead to alteration or loss of the genetic information encoded in this vitally important molecule. Known causes of mtDNA instability include reactive oxygen species, radiation and toxic chemicals, but also replication errors that can arise from e.g. defects in the machinery that catalyzes mtDNA replication or a shortage or imbalance of the DNA building blocks, dNTPs (Figure 1).


Figure 1. A simplified overview of mtDNA instability. Various forms of insults and stresses (in blue) can damage or alter the genetic information encoded in the mtDNA by introducing e.g. strand breaks, oxidized bases or mismatches. MtDNA instability is associated with numerous forms of human disease. (Image created using BioRender.)

The major aim of our work is to increase our understanding of the array of mechanisms that either prevent, or help cope with, various forms of mtDNA instability. Our research projects address an array of questions related to mtDNA instability, ranging from the requirements for faithful mtDNA replication and the effects of incorporated ribonucleotides to the cellular consequences of mtDNA instability.

Requirements for faithful mtDNA replication

MtDNA replication is carried out by a dedicated replication machinery that consists of the mitochondrial DNA polymerase ɣ, the TWINKLE helicase and the mitochondrial single-stranded DNA-binding protein mtSSB. Defects in any of these factors of the core replisome can result in mtDNA instability in the form of mtDNA depletion, mutations or large deletions, and consequently human disease 1. In addition to the functional replisome, mtDNA maintenance also requires a sufficient and balanced supply of dNTPs. A part of our research will help define the requirements for faithful mtDNA replication in terms of both protein factors and the dNTP supply.

Effect of ribonucleotides on mtDNA stability

A peculiar feature of mtDNA is that it contains incorporated ribonucleotides (rNMPs), building blocks that are normally used to build RNA but occasionally get incorporated into DNA during the replication process. In contrast to the nucleus that contains a dedicated repair pathway for the removal of rNMPs incorporated in the genome 2, mitochondria lack efficient rNMP repair and these building blocks therefore persist in the mtDNA 3.

The presence of rNMPs in DNA is known to increase the risk of strand breaks, and affects the structure and elasticity of the DNA 4-6. Despite the well-established adverse effect of rNMPs on the stability of the nuclear genome, we have shown that the physiological level of mtDNA rNMPs is well-tolerated and does not appear to have obvious effects on mtDNA stability in vivo 7. However, the frequency and identity of rNMPs is altered in cultured cells or mice carrying disease-associated mutations in enzymes of nucleotide metabolism 8,9. It is thereby possible that e.g. an increased mtDNA rNMP load in mitochondrial disease could influence mtDNA-dependent processes. We aim to better understand the mechanisms underlying the differential effects of rNMPs on mtDNA vs. the nuclear genome.  

Consequences of mtDNA instability

In recent years, it has become increasingly clear that mitochondria are not isolated organelles in the cytosol but rather central hubs of cellular metabolism that are well-integrated with other components in the cell. Mitochondria play roles in many processes in addition to energy production, such as nucleotide or amino acid synthesis, apoptosis, Fe-S cluster synthesis and Ca2+ homeostasis, to name a few. They are in constant dialogue with other components of the cell such as the nucleus, ER as well as lysosomes using a variety of different signals (summarized in e.g. 10). Therefore, changes in mitochondrial function, such as decreased mitochondrial energy production, are conveyed to the rest of the cell and trigger adaptive responses that are aimed at restoring normal function, be it by boosting mitochondrial biogenesis, clearing away dysfunctional mitochondria through mitophagy, or even delaying cell proliferation until sufficient mitochondrial function is restored 10,11. However, mito-cellular communication is involves multiple interconnected signaling pathways, and although new knowledge accumulates at a rapid rate, we still lack a comprehensive understanding of the means and outcomes of this complex communication network.

A major focus of our work aims to address the types of signals and the cellular consequences that are triggered upon mtDNA instability. We use two complementary biological systems in our studies: baker’s yeast Saccharomyces cerevisiae as well as cultured human cells in order to facilitate the identification of signals and factors involved in mito-cellular communication. Furthermore, we make use of our expertise in biochemistry to study the mechanistic details of this communication using purified recombinant proteins in relevant in vitro assays.

References

  1. Doimo, M., Pfeiffer, A., Wanrooij, P. H. & Wanrooij, S. in The Human Mitochondrial Genome (eds. Gasparre, G. & Porcelli, A. M.) 3–33 (Academic Press, 2020). doi:10.1016/B978-0-12-819656-4.00001-2
  2. Sparks, J. L. et al. RNase H2-initiated ribonucleotide excision repair. Mol Cell 47, 980–986 (2012).
  3. Wanrooij, P. H. et al. Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired. Proc. Natl. Acad. Sci. U.S.A. 114, 12466–12471 (2017).
  4. Jaishree, T. N., van der Marel, G. A., van Boom, J. H. & Wang, A. H. Structural influence of RNA incorporation in DNA: quantitative nuclear magnetic resonance refinement of d(CG)r(CG)d(CG) and d(CG)r(C)d(TAGCG). Biochemistry 32, 4903–4911 (1993).
  5. Chiu, H.-C. et al. RNA intrusions change DNA elastic properties and structure. Nanoscale 6, 10009–10017 (2014).
  6. Li, Y. & Breaker, R. R. Kinetics of RNA degradation by specific base catalysis of transesterification involving the 2γ-hydroxyl group. Journal of the American Chemical Society 121, 5364–5372 (1999).
  7. Wanrooij, P. H. et al. Elimination of rNMPs from mitochondrial DNA has no effect on its stability. Proceedings of the National Academy of Sciences 117, 14306–14313 (2020).
  8. Berglund, A.-K. et al. Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA. PLoS Genet 13, e1006628 (2017).
  9. Moss, C. F. et al. Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model. Nucleic Acids Res 45, 12808–12815 (2017).
  10. Mottis, A., Herzig, S. & Auwerx, J. Mitocellular communication: Shaping health and disease. Science 366, 827–832 (2019).
  11. Martínez-Reyes, I. et al. TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions. Mol Cell (2015). doi:10.1016/j.molcel.2015.12.002