Classification of topoisomerases

1.5 DNA TOPOLOGY

1.5.2 Classification of topoisomerases

The key feature for all topoisomerases is to contain a nucleophilic tyrosine and to form a covalent phosphotyrosyl linkage between the phosphate group in the DNA backbone via nucleophilic attack of the active site of tyrosine.

Topoisomerases are classified as type I and II based on this strand scission activity, in which type I enzymes catalyze the formation of a single-stranded break (Depew et al., 1978; Tse et al., 1980) and type II enzymes cleave both strands (Wang, 1998; Champoux, 2001; Bush et al., 2015). The human genome encodes six topoisomerases (TOP1, TOP1MT, TOP2A, TOP2B, TOP3A and

1.4.6 Mitochondrial diseases

Mitochondrial diseases are a clinically heterogeneous group of diseases caused by a chronic failure of ATP production through defective OXPHOS. These diseases primarily affect tissues with high energy demand, such as skeletal muscle, cardiac muscles, extraocular muscles, brain, liver, nervous system.

Clinical presentations of mitochondrial diseases can vary and include progressive external ophthalmoplegia, ptosis, heart and kidney failure, deafness, diabetes, parkinsonism, male infertility, spasticity, dysmotility, and obesity (Chan & Copeland, 2009; Greaves et al., 2012; Kanungo et al., 2018).

Mitochondrial dysfunction has also been associated with the biological ageing process aging (Trifunovic et al., 2004; Kauppila et al., 2017). Mitochondrial diseases may arise in different ways: via mutations/rearrangements of mtDNA itself or mutations in nuclear-encoded mitochondrial proteins involved in mitochondrial maintenance (Copeland, 2012; Copeland & Longley, 2014).

Human mitochondrial diseases resulting from mtDNA mutations are maternally inherited since mtDNA is passed on only from the oocyte. The multicopy nature of mtDNA allows the existence of wild-type and mutant mtDNA molecules as a mixed population in the cells, which is known as heteroplasmy, and there is a threshold level of genomes containing the mutations that must be passed before a phenotype is observed in cells (Stewart

& Chinnery, 2015). The vast majority of mtDNA mutations are considered functionally recessive (Lax et al., 2011), even though some dominant mutations have been reported (Sacconi et al., 2008). Mitochondrial mutations can be categorized based on (i) mutations affecting OXPHOS, (ii) mutations affecting mtDNA metabolism (replication, expression, maintenance), (iii) mutations affecting mitochondrial dynamics and (iv) mutations affecting mitochondrial membranes. The prevalence of mitochondrial-related disorders has been estimated to be greater than 1 in 5,000 individuals (Ng & Turnbull, 2016). Mitochondrial diseases can caused by mutations in large number of nuclear genes, such as the DNA polymerase POLg, the replicative helicase TWINKLE, the RNA polymerase POLRMT, the nucleases MGME1 and RNaseH1, and the topoisomerase TOP3A (Spelbrink et al., 2001; Kornblum et al., 2013; Copeland, 2014; Reyes et al., 2015; Oláhová et al., 2021; Primiano et al., 2022). In paper I and paper II of this thesis we discuss novel mutations in POLg and TOP3A associated with mitochondrial disease.

1.5 DNA TOPOLOGY

The topology of DNA changes during replication, transcription, and repair.

Hence, mechanisms are required to modulate DNA topology, and a group of enzymes known as topoisomerases perform these vital roles (Vos et al., 2011).

1.5.1 Why do we need topoisomerases?

DNA is a double-stranded helix molecule in which the two strands are wound around one another, a structure that would be very challenging for living organisms if topoisomerases did not exist. For example, during DNA replication, DNA strands must be separated, which causes the formation of positive supercoils (DNA overwinding) ahead of the replication fork and negative supercoils (underwinding) behind it. Unresolved positive supercoiling can inhibit the progression of the replication fork. Similarly, during transcription, positive and negative supercoiling are generated by RNA polymerase (Liu & Wang, 1987). Additionally, DNA repair generates entanglements between DNA regions that can cause potentially mutagenic or cytotoxic DNA strand breaks if they are left unresolved. For these reasons, topoisomerases are essential enzymes for all organisms; they can allow the cell to efficiently replicate, transcribe, and maintain genome integrity by manipulating DNA supercoiling (Bates &Maxwell 2005; McKie et al., 2021).

The basic principle of topoisomerases is to create a transient break in the DNA backbone, which allows DNA to untwist and untangle before the break is resealed (Alberts, 2008; Deweese et al., 2009; Vos et al., 2011). Although all topoisomerases perform a similar task, which is releasing topological stress, the mechanism of this achievement varies between enzyme classes.

1.5.2 Classification of topoisomerases

TOP3B) belonging to the subgroups type IA (TOP3A and TOP3B), type IB (TOP1, TOP1MT) and type IIA (TOP2A, TOP2B). Type IA topoisomerases cleave the DNA backbone, generating a covalent linkage to the 5′-phosphate function via a strand passage mechanism, whereas type IB topoisomerases cleave the DNA backbone, generating a covalent linkage to the 3′-phosphate and function via a controlled rotation mechanism (Stewart et al., 1998). Both type IA and type IB topoisomerases catalyze single-stranded breaks. However, type IIA topoisomerases are different since they are able to cleave both strands of a DNA duplex and create a double-stranded break through which they pass a second duplex (Figure 7). Additionally, in contrast to type IA and IB topoisomerases, type IIA topoisomerases use ATP to drive strand passage (Goto & Wang, 1982).

Figure 7. Overview of topoisomerases and their working principles. Type I topoisomerases introduce single cut, whereas Type II topoisomerases cut two strands of double-stranded DNA. The image was created with BioRender.

Different catalytic properties of topoisomerases allow them to work on different substrates with different mechanisms. For example, a controlled rotation mechanism allows type IB topoisomerases to relax positive and negative supercoils, whereas the strand passage mechanism of type IA leads to the relaxation of negative supercoils and decatenate single-stranded DNA molecules. Moreover, type IIA topoisomerases can relax positive and negative

supercoils and decatenate interlinked double-stranded DNA molecules (Nitiss, 2009).

1.5.2.1 TOP1

There are two paralogous genes encoding type IB topoisomerases in human cells, TOP1 localized to the nucleus and TOP1MT with a mitochondrial localization. It is believed that invertebrates have only a single form of TOP1, which functions in both nuclei and mitochondria (Dalla Rosa et al., 2009);

however, it is not clear why vertebrates require two distinct paralogs of enzymes dedicated to either organelle. This may be because the transcription and replication machineries acting on mtDNA are significantly different between yeast and mammals (Falkenberg et al., 2007). TOP1 is a key nuclear enzyme that is able to change DNA topology and has ubiquitous roles in important cellular functions such as DNA replication, recombination, and transcription (Baranello et al., 2016; Kim & Jinks-Robertson, 2017). TOP1 is essential for early development in mice, and enzyme expression is required very early after fertilization (Morham et al., 1996).

1.5.2.2 TOP2A and TOP2B

There are two type IIA topoisomerases in human cells, TOP2A and TOP2B, which play different roles in nuclear DNA maintenance and expression.

TOP2A is highly expressed in rapidly dividing cells such as pluripotent embryonic stem cells (Thakurela et al., 2013). In the nucleus, TOP2A is required for resolving DNA bridges during anaphase and is an essential enzyme for chromosome segregation (Nielsen et al., 2020), whereas TOP2B has a primary role in differentiation, maturation and neural development (Austin et al., 2018). The mitochondrial localization of these two proteins is an active area of debate. Previous studies have suggested that both TOP2 isoforms are present in mitochondria in human cells. Zhang et al., (2014) identified TOP2A and the full-length form of TOP2B in mitochondria. Moreover, another study suggested that a truncated form of TOP2B was found in mitochondria (Low et al., 2003). However, more recent studies have failed to detect any of these type IIA topoisomerases in mitochondria (Nicholls et al., 2018; Menger et al., 2022). In paper III, we show that neither of the two types IIA topoisomerases, TOP2A and TOP2B, has a detectable mitochondrial targeting sequence, in contrast to TOP3A and TOP1MT, so it remains unclear how their mitochondrial import would be achieved. In addition to the presence

supercoils and decatenate interlinked double-stranded DNA molecules (Nitiss, 2009).

1.5.2.1 TOP1

1.5.2.2 TOP2A and TOP2B

There are two type IIA topoisomerases in human cells, TOP2A and TOP2B, which play different roles in nuclear DNA maintenance and expression.

of targeting sequences belonging to TOP3A and TOP1MT, the mitochondrial localization of these two proteins is supported by proteomic and bioinformatic studies (Rhee et al., 2013; Calvo et al., 2016).

1.5.2.3 TOP3B

TOP3B is unique among the human topoisomerases since it is able to process both RNA and DNA substrates (Ahmad, 2017). TOP3B localizes to both the cytosol and the nucleus, but not to mitochondria. Although it is not essential, mice ablated for TOP3B show a shortened lifespan, infertility, an increased rate of aneuploidy in germ cells and abnormal synapse formation (Kwan &

Wang, 2001; Xu et al., 2013). It has also been reported that individuals with TOP3B deletion suffer from neurodevelopmental disorders that are related to the RNA topoisomerase activity of TOP3B (Stoll et al., 2013). TOP3B forms a complex with TDRD3, which targets TOP3B to resolve R-loop structures associated with nuclear transcription (Siaw et al., 2016). Cytosolic TOP3B is associated with the translation of mRNAs together with RNA binding proteins (RBPs), which are important for neurodevelopment and mental health (Ahmad et al., 2017). TOP3B is necessary for preventing excessive R-loop formation and loss of TOP3B increases DNA damage and genome instability (Zhang et al., 2019).

No documento mitochondrial DNA maintenance and topology (páginas 34-38)