Topoisomerases and mitochondrial diseases

Mutations in topoisomerases can cause human diseases associated with different clinical manifestations, ranging from developmental delay to autism, Bloom syndrome, mitochondrial diseases, premature aging, and carcinogenesis (Nicholls et al., 2018; Martin et al., 2018; Zhang et al., 2019;

Lee & Wang, 2019; Pommier et al., 2022).

Mutations in the TOP3A gene cause mtDNA deletion, impaired mtDNA segregation and ultimately mtDNA aggregation (Nicholls et al., 2018). The clinical symptoms and molecular features of these mutations are similar to those associated with some disease causing mutations in other proteins, such as components of the mtDNA replication machinery (Viscomi & Zeviani, 2017). Another cohort of individuals with biallelic TOP3A mutations displayed symptoms similar to those seen in Bloom syndrome, including prenatal onset growth restriction and microcephaly (Martin et al., 2018).

Interestingly, Bloom syndrome is caused by mutations in the gene encoding the BLM helicase, which interacts with TOP3A in the BTTR complex. Several of the patients with the Bloom-like syndrome also displayed mitochondrial dysfunction, including cardiomyopathy, mtDNA depletion in muscles and progressive external ophthalmoplegia (PEO), which were likely caused by the loss of mitochondrial TOP3A activity (Martin et al., 2018). More recently, another study reported that two siblings with two novel mutations in TOP3A exhibited disorders similar to Bloom syndrome with cardiomyopathy and mitochondrial dysfunction, such as defective mitochondrial respiration (Jiang et al., 2021). Moreover, in this thesis, in paper II, we characterize nine different pathological TOP3A variants causing adult-onset mitochondrial diseases and Bloom-like syndrome.

Disease causing mutations have not yet been identified in the gene encoding TOP1MT. However, one study characterized two single nucleotide variants in TOP1MT, which were located in highly conserved regions of the protein across species and affected the protein’s catalytic activity (Zhang et al., 2017).

Further investigations are warranted to elucidate the possible relevance of TOP1MT mutations as a source of mitochondrial diseases and potential therapies.

possible for cancer cells to grow in an environment with low levels of oxygen and nutrients (Baechler et al., 2019).

1.5.3.2 TOP3a (TOP3A)

Another mitochondrial type I isomerase is Top3a (TOP3A), which has both a nuclear and mitochondrial localization in mammalian cells. Mitochondrial TOP3A protein is produced from the same gene (TOP3A) as its nuclear counterpart (Wang et al., 2002; Nicholls et al., 2018; Nicholls & Gustafsson, 2018). Translation initiation from the first ATG yields a mitochondrial isoform of TOP3A, whereas translation from a downstream ATG generates a nuclear isoform of TOP3A lacking the mitochondrial targeting sequence (Wang et al., 2002). How this alternative translation is controlled is unclear, but it is clear that proper targeting of TOP3A is critical and that the mistargeting of TOP3A can be a possible reason for human diseases associated with mtDNA depletion (Wu et al., 2010). TOP3A is essential for early development both in mouse and Dropsophila (Li & Wang, 1998; Plank et al., 2005), and in the fly, the absence of TOP3A causes an increase in mtDNA deletions and decrease in mtDNA copy number by at least 75% and (Wu et al., 2010 ; Tsai et al., 2016). Recently, it was shown that the TOP3A protein is essential for mtDNA separation in mammalian cells (Nicholls et al., 2018). TOP3A has a critical role in the decatenation of newly synthesized mtDNA molecules after replication termination. In the same study, they demonstrated that a loss of TOP3A causes the formation and accumulation of catenated networks of mtDNA, similar to mtDNA dimers characterized almost 60 years ago (Hudson & Vinograd, 1967;

Nicholls et al., 2018). These termination structures are defined as hemicatenanes, that is, double-stranded DNA (dsDNA) molecules associated through a single-stranded (ss) linkage (Nicholls et al., 2018). The presence of hemicatenane structures is consistent with the catalytic activity of TOP3A, as a member of the type IA topoisomerase family, which uses an enzyme-bridge strand passage mechanism allowing it to act as an ss decatenase and relieve negative supercoiling (Nicholls et al., 2018). The nuclear isoform of TOP3A forms a complex with the ReQ family helicase BLM and OB-fold proteins RMI1 and RMI2, which is called the BTRR complex (Wu & Hickson, 2003;

Xu et al., 2008; Singh et al., 2008). The role of the BTRR complex is to catalyze the dissolution of double-Holliday junctions during homologous recombination and produce noncrossed-over products (Raynard et al., 2008).

Recently, it has been reported that the DNA translocase PICH cooperates with nuclear TOP3A/RMI1/RMI2 (TRR complex) to induce positive DNA supercoiling (Bizard et al., 2019), and this cooperation facilitates the

decatenation activity of TOP3A at the onset of anaphase (Baxter et al., 2011).

It is important to highlight that these nuclear binding partners of TOP3A have not been observed to localize to mitochondria, and it remains unknown whether mitochondrial TOP3A has any interaction partners (Nicholls et al., 2018).

1.5.4 Topoisomerases and mitochondrial diseases

Mutations in topoisomerases can cause human diseases associated with different clinical manifestations, ranging from developmental delay to autism, Bloom syndrome, mitochondrial diseases, premature aging, and carcinogenesis (Nicholls et al., 2018; Martin et al., 2018; Zhang et al., 2019;

Lee & Wang, 2019; Pommier et al., 2022).

Mutations in the TOP3A gene cause mtDNA deletion, impaired mtDNA segregation and ultimately mtDNA aggregation (Nicholls et al., 2018). The clinical symptoms and molecular features of these mutations are similar to those associated with some disease causing mutations in other proteins, such as components of the mtDNA replication machinery (Viscomi & Zeviani, 2017). Another cohort of individuals with biallelic TOP3A mutations displayed symptoms similar to those seen in Bloom syndrome, including prenatal onset growth restriction and microcephaly (Martin et al., 2018).

Interestingly, Bloom syndrome is caused by mutations in the gene encoding the BLM helicase, which interacts with TOP3A in the BTTR complex. Several of the patients with the Bloom-like syndrome also displayed mitochondrial dysfunction, including cardiomyopathy, mtDNA depletion in muscles and progressive external ophthalmoplegia (PEO), which were likely caused by the loss of mitochondrial TOP3A activity (Martin et al., 2018). More recently, another study reported that two siblings with two novel mutations in TOP3A exhibited disorders similar to Bloom syndrome with cardiomyopathy and mitochondrial dysfunction, such as defective mitochondrial respiration (Jiang et al., 2021). Moreover, in this thesis, in paper II, we characterize nine different pathological TOP3A variants causing adult-onset mitochondrial diseases and Bloom-like syndrome.

Disease causing mutations have not yet been identified in the gene encoding TOP1MT. However, one study characterized two single nucleotide variants in TOP1MT, which were located in highly conserved regions of the protein across species and affected the protein’s catalytic activity (Zhang et al., 2017).

Further investigations are warranted to elucidate the possible relevance of TOP1MT mutations as a source of mitochondrial diseases and potential therapies.

2 AIMS

In this thesis we aim to gain biochemical insight into mitochondrial DNA maintenance and topology. In the four papers of this thesis, we have addressed different questions regarding the mechanisms of these processes. Our goal also is to understand the molecular mechanisms of disease-causing mutations that affect these processes and cause clinical symptoms in patients.

The specific aims for each papers are:

Paper I: To characterize a novel disease-causing mutation (F907I) in POLg, to elucidate how this amino acid substitution disturbs mtDNA replication in affected patients.

Paper II: To characterize new pathological variants in TOP3A causing distinct disorders of mitochondrial and nuclear genome stability.

Paper III: To investigate the roles of two type I topoisomerases (TOP3A and TOP1MT) in maintaining DNA topology in human mitochondria

Paper IV: To characterize the activity and submitochondrial localization of human mitochondrial EXOG

2 AIMS

In this thesis we aim to gain biochemical insight into mitochondrial DNA maintenance and topology. In the four papers of this thesis, we have addressed different questions regarding the mechanisms of these processes. Our goal also is to understand the molecular mechanisms of disease-causing mutations that affect these processes and cause clinical symptoms in patients.

The specific aims for each papers are:

Paper I: To characterize a novel disease-causing mutation (F907I) in POLg, to elucidate how this amino acid substitution disturbs mtDNA replication in affected patients.

Paper II: To characterize new pathological variants in TOP3A causing distinct disorders of mitochondrial and nuclear genome stability.

Paper III: To investigate the roles of two type I topoisomerases (TOP3A and TOP1MT) in maintaining DNA topology in human mitochondria

Paper IV: To characterize the activity and submitochondrial localization of human mitochondrial EXOG

3 RESULTS