Replication stress and damage tolerance in mammalian mitochondria

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ISBN 978-952-61-2887-0 ISSN 1798-5668

Dissertations in Forestry and Natural Sciences

DISSERTATIONS | RUBÉN TORREGROSA MUÑUMER | REPLICATION STRESS AND DAMAGE TOLERANCE... | No 315

RUBÉN TORREGROSA MUÑUMER

REPLICATION STRESS AND DAMAGE TOLERANCE IN MAMMALIAN MITOCHONDRIA

PUBLICATIONS OF

THE UNIVERSITY OF EASTERN FINLAND

Mitochondria are popularly known as ”the power houses of the cells”. Interestingly, they contain their own genome,

the mitochondrial DNA (mtDNA). How mammalian mtDNA is copied is still under debate. This doctoral thesis

provides data on how mammalian mtDNA is replicated under steady-state conditions and under replication stress (when the DNA template is damaged). It seems that

mammalian mitochondria have two different replication mechanisms, which are differently used depending on the reining conditions. In addition, this thesis also investigates how mammalian mitochondria are able to endorse different replication restart strategies, including

recombination-mediated repair and repriming.

RUBÉN TORREGROSA MUÑUMER

30874055_UEF_Vaitoskirja_NO_315_Ruben_Torregrosa_Muñumer_Lumet_cover_18_09_07.indd 1 7.9.2018 13.06.29

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REPLICATION STRESS AND DAMAGE TOLERANCE IN MAMMALIAN

MITOCHONDRIA

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Rubén Torregrosa Muñumer

REPLICATION STRESS AND DAMAGE TOLERANCE IN MAMMALIAN

MITOCHONDRIA

Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences

No 315

University of Eastern Finland Joensuu

2018

Academic dissertation

To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium F101 in the Futura Building at the University of Eastern Finland, Joensuu, on 28^th of September, 2018, at

12 o’clock noon

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Grano Oy Jyväskylä, 2018 Editors: Matti Vornanen

Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto

ISBN: 978-952-61-2887-0 (Print) ISBN: 978-952-61-2888-7 (PDF)

ISSNL: 1798-5668 ISSN: 1798-5668 (Print)

ISSN: 1798-5676 (PDF)

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Author’s address: Rubén Torregrosa Muñumer University of Eastern Finland

Depart. of Environmental and Biological Sciences P.O. Box 111

80101 JOENSUU, FINLAND email: ru.torregrosa@gmail.com

Supervisors: Jaakko Pohjoismäki, Ph.D.

University of Eastern Finland

80101 JOENSUU, FINLAND email: jaakko.pohjoismaki@uef.fi

Steffi Goffart, Ph.D.

University of Eastern Finland

80101 JOENSUU, FINLAND email: steffi.goffart@uef.fi

Reviewers: Principal investigator Michal Minczuk, Ph.D.

MRC Mitochondrial Biology Unit (MRC MBU) University of Cambridge

Wellcome Trust/MRC Building

Cambridge Biomedical Campus, Hills Road CB2 0XY CAMBRIDGE, UNITED KINGDOM

email: michal.minczuk@mrc-mbu.cam.ac.uk Senior Investigator ScientistAurelio Reyes, Ph. D.

MRC Mitochondrial Biology Unit (MRC MBU) University of Cambridge

Wellcome Trust/MRC Building

Cambridge Biomedical Campus, Hills Road CB2 0XY CAMBRIDGE, UNITED KINGDOM email:art@mrc-mbu.cam.ac.uk

Opponent: Professor Howard T Jacobs University of Tampere

Faculty of Medicine and Life Sciences FI-33014 TAMPERE, FINLAND email: howard.t.jacobs@uta.fi

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7 Torregrosa Muñumer, Rubén

Replication stress and damage tolerance in mammalian cell mitochondria Joensuu: University of Eastern Finland, 2018

Publications of the University of Eastern Finland

Dissertations in Forestry and Natural Sciences 2018; No 315 ISBN: 978-952-61-2887-0 (Print)

ISSNL: 1798-5668 ISSN: 1798-5668 (Print) ISBN: 978-952-61-2888-7 (PDF) ISSN: 1798-5676 (PDF)

ABSTRACT

Mitochondria are tiny organelles inside most eukaryotic cells. They provide the energy for the cells to function and therefore are essential especially in highly metabolic tissues such as the heart or brain. Mitochondria contain their own DNA, the mitochondrial DNA (mtDNA), which must be replicated to be distributed between daughter cells or respond to rising energy demands. Moreover, mitochondria are thought to harbor an oxidative environment as a trade-off for their energy production. Damage on the mtDNA can impair replication and cause replication stalling, which can in turn lead to mtDNA genomic instability and mitochondrial diseases. These diseases are relatively common forms of neuromuscular disorders, affecting between 1:7,000 and 1:20,000 individuals under 16 years of age. Although extremely relevant, the diagnosis, prognosis and treatment of mitochondrial diseases are challenging due to their clinical and genetic heterogeneity. In spite of the important role that mtDNA plays in these disorders, little is known about its maintenance. For example, at least three different models of mtDNA replication have been proposed, including asynchronous, strand-coupled and recombination-mediated replication, and almost nothing is known about how mtDNA replication deals with a damaged DNA template (i.e., replication stress).

This study investigates how mammalian mtDNA is replicated under different conditions and how mtDNA replication copes with replication stress. To address these questions, I used several animal cell lines which were exposed to different DNA-damaging agents, including UV light, KBrO³ (an inducer of oxidative damage) and dideoxycytidine (ddC, a specific inhibitor of mtDNA replication). These agents are expected to cause lesions on the mtDNA template or directly block replication, inducing artificial replication fork stalling and replication stress. If stalled forks are not rescued, they can finally collapse, inducing double-strand breaks and prompting mtDNA genetic instability. In prokaryotes and the eukaryotic nucleus, several options exist to rescue stalled forks, including recombination, fork regression and

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repriming. Mainly combining one-dimension and two-dimension agarose gel electrophoresis, I analyzed how mtDNA replication proceeds under replication stress. Moreover, to evaluate the possible role of several candidate proteins in replication stress, I used different cell lines lacking the gene of interest (knockout) or overexpressing wild-type or mutant versions of the given protein. With these tools, I studied the possible participation of the helicase TWNK and exonuclease MGME1 in fork regression and recombination, as well as the role of the polymerase/primase Primpol in repriming downstream of the lesion.

The study’s findings support the idea of at least two replication mechanisms co- existing in mammalian mitochondria. Under normal conditions mtDNA is duplicated following an asynchronous replication mechanism, while a strand- coupled or synchronous replication is preferred under replication stress. The latter is accompanied by the accumulation of fully dsDNA cruciform molecules (recombination intermediates and regressed forks), suggesting that, as in prokaryotes and the eukaryotic nucleus, these strategies are pursued in mammalian mitochondria to resolve stalled forks. To understand the formation and processing of these structures, I focused on two protein candidates expected to be involved in fork rescue: the helicase TWNK and the exonuclease MGME1. Although mitochondrial recombination is still uncertain, TWNK is a good candidate to be the missing mitochondrial recombinase as it can induce strand exchange reactions in vitro and is connected to the induction of recombination in animal models and the human heart. On the other hand, patients with deficient MGME1 accumulate mtDNA rearrangements and linear deletions, suggesting a possible role in the processing of stalled forks. Manipulation of both TWNK and MGME1 proteins resulted in the modification of cruciform forms. Although currently not fully conclusive, these data suggest that TWNK may be involved in recombination- mediated repair. On the other hand, as previously suggested, my data support the role of MGME1 in the formation of mtDNA deletions and rearrangements through its involvement in the processing of DNA flaps and the turnover of linear fragments, although other functions in mtDNA replication cannot be discarded. Moreover, this study supports the hypothesis that mtDNA deletion and rearrangement formation are linked to replication stalling.

Another strategy to rescue stalled forks is repriming downstream of the blocking lesion, allowing replication to resume. The polymerase/primase Primpol has recently been characterized in the eukaryotic nucleus and mitochondria. Although Primpol may also act as a translesion (TLS) polymerase, inserting a nucleotide opposite of the lesion, the literature rather supports a role in repriming. While this role has been proposed for mammalian mitochondria, it has not been experimentally addressed.

This study’s findings indicate that Primpol indeed also acts as a primase in mitochondria, rescuing stalled replication forks by repriming downstream. In

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9 addition, artificial manipulation of mitochondrial Primpol levels resulted in the conversion of partially single-stranded DNA (the signature of asynchronous replication) to fully double-stranded DNA (a feature of strand-couple replication).

This suggests that Primpol might prime at non-canonical sites and therefore may be involved in the lagging-strand synthesis during strand-coupled replication.

In sum, these findings help to better understand how mtDNA is maintained, especially under replication stress conditions. The more extensive knowledge of the molecular events governing mtDNA maintenance can also be expected to help diagnose mitochondrial diseases, as well as in their prognosis and possible treatment.

CAB Thesaurus: mitochondrial DNA, mitochondrial genetics, DNA replication

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"Observar sin pensar es tan peligroso como pensar sin observar"

Santiago Ramón y Cajal

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ACKNOWLEDGEMENTS

Los primeros agradecimientos van dirigidos a mi familia, sin cuyo apoyo y cariño jamas habría sido capaz de acabar la carrera; y aún más importante, empezar un doctorado en otro país. A vosotros, mis padres, os debo en gran medida mi devoción por la ciencia. Aunque al final me decanté por los seres vivos más que por las rocas, vosotros me inculcásteis el afán por querer saber más; tanto a través de los libros, como disfrutando de la naturaleza. Gracias por alimentar mi curiosidad. Marcos, tu te mereces unas líneas a parte por todo el trabajo de edición de los gráficos y figuras que elegantemente ilustran esta tesis. Y junto a Noe, gracias por esa visita que me hicísteis. Cuando crezca un poco más el pequeñajo le podréis leer esta tésis para que se duerma.

Cronológicamente empezaré agradeciendo la oportunidad que se me concedió cuando como estudiante de carrera empecé a tener mis primeros contactos con la investigación en la Unidad de Investigación de La Paz. Muchas gracias Mayte por hacerme un hueco en tu grupo y empezar a modelar mis habilidades en el laboratorio. Eduardo, muchas gracias por motivarme e inspirarme, y por esas discusiones científicas en las que me iniciaste. Durante mi periplo por la Facultad de Medicina de la Universidad Complutense de Madrid tuve la oportunidad de conocer gente extraordinaria, incluyendo Raquel, Ernesto, Maria (s), Bea, Sandra y Ricardo.

Muchísimas gracias por toda la cienca, cafés (y demás) y vuestro apoyo. Ricardo, muchísimas gracias por tu ayuda, por lo que me enseñaste y por descubrirme el mundo del mantenimiento del ADN mitochondrial. Así mismo, gracias Victoria, Vicente, Elena, Jesús y Roman por vuestra ayuda y apoyo.

My next step placed me in Finland, where I had the chance to meet amazing people who in different levels helped me in my way trough my PhD and new life in this amazing country. In first place I would like aknowledge my supervisors, Steffi and Jaakko. Thanks for giving me such an opportunity, for your hospitality, for all the technical support, the science and everything you have taught me. Nina, thanks for being there during the first steps of my PhD and showing me how it is. Anu, thank you very much for all those moments we shared inside and outside the lab.

Thanks for the balance and the discussions. To my students: Juha, Jukka, Obaidur and Karolina, thanks for everything you taught me. And thank you very much to the Department of E. and Biological sciences at UEF. You became part of my family during these years! Thanks Matti Vornanen for all your help during the publishing process. And thank you very much, Henna, for giving me the opportunity to continue doing science in your lab.

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Special thanks to Sjoerd, Sonja, Josefin, Annika, Mara, Gorazd, Kazu and Valentin for hosting me and making me feel like at home during my visit to Umeå. Sjoerd, special thanks for the science and the technical support. Thanks you all for your great contribution to the Primpol story. Big thanks to Blanco’s group for your very valuable contribution to the Primpol work. Luis, thank you very much for the long phone conversations and mentoring. In addition, thanks to Wolfram’s group for your great contribution to the MGME1 story.

Thanks to Howard T. Jacobs. It will be a great honor to count on one of the most important contributors to mtDNA maintenance as my opponent. And thanks to the pre-examiners, Arturo and Michal, for reviewing and commenting on this quite large thesis.

Big thanks to Ilari, Sari, Kimo and Kaisa. Thanks for becoming my “Finnish family” and sharing with me many things about the Finnish culture, which would have passed unnotticed to me. Thanks for your support! Kiitos paljon. Olalla y Luis, muchas gracias por ser como sois, por vuestra compañía y por esos increíbles viajes que nos hemos marcado. Habrá que repetir y quedarse a dormir en algún mostal.

Gracias Lomeros! Juha and Isa, thanks for being there when needed and for all those moments we spent together in Joensuu and Lapland. Albert, muchas gracias por esas largas conversaciones y por todos esos momentos (también esos de “una y nos vamos”). And thanks to Pirkko, Javier, Ulla, Blanca, Jussi, Yasemin, Jani, Mar, Antonio, Adrián, Aitor, Blas, Jaume, Seija, Cheikh, German and Gorgo for your support. Leo, Juha, Niko and others from Joensuu Airsoft ry and Fantasiapelit, thanks for all those moments in the battlefield. Miia(s), Eija, Maija, Joni, Kari and other people from Salsa del Este, thanks for helping me to disconnect from the lab.

Gracias a los chicos de Modelbrush por esa constancia y por la increible visita que os marcásteis en aquellos momentos de flojera. Espero que tuviéseis suficiente nieve. Y gracias a la Comunidad del Anillo, por estar siempre ahí con los brazos abiertos durante mis vacaciones por España.

Elena, thank you very much for being the way you are. Thanks for everything you know, specially for your support and patience during the last and stressful months of my PhD. Вы были светом в конце туннеля 

Thanks to Jane & Aatos Erkko Foundation for your support. Finally, special thanks to my cell lines. I will never forget all the moments we spent together, including those in the middle of the night in the worst part of the winter. And thank you, mitochondria, for providing me with enough energy to finish this thesis.

Helsinki, August 2018 Rubén Torregrosa Muñumer

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LIST OF ABBREVIATIONS

2D-AGE Two-Dimensional Agarose Gel Electrophoresis AEP Archeo-Eukaryotic Primase

ATP Adenosine triphosphate ADP Adenosine diphosphate ddC 2,3- dideoxycytidine dsDNA double-stranded DNA BER Base Excision Repair BIR Break-Induced Repair

bp base pair

CPD Cyclobutane Pyrimidine Dimer CSB Conserved Sequence Block

COSCOFA Conventional Strand-Coupled Okazaki Fragment Associating DMEM Dulbecco´s Modified Eagle Medium

DNA Deoxyribonucleic Acid

dNTP Deoxynucleoside triphosphate DSB Double-Strand Break

ER Endoplasmic Reticulum EtBr ethidium bromide ETC Electron Transport Chain

FADH² Reduced Flavin Adenine Dinucleotide GC Gene Conversion pathway

GpC Gapped Circle

HEK 293 Human Embryonic Kidney cell line HR Homologous Recombination HJ Holiday junction

HSP Heavy-Strand Promoter (transcription) kDa Kilo Dalton

LSP Light-Strand Promoter (transcription) LTR Long Terminal sequences

MEF mouse embryonic fibroblast mtDNA mitochondrial DNA

MTS mitochondrial targeting sequence

NAD⁺ oxidized Nicotinamide Adenine Dinucleotide NADH reduced Nicotinamide Adenine Dinucleotide NCR Non-Coding Region

nDNA nuclear DNA

NER Nucleotide Excision Repair pathway

nt nucleotide

OL Light (lagging)-Strand Origin of Replication OH Heavy (leading)-Strand Origin of Replication

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Ori-z Initiation zone of bidirectional replication OXPHOS Oxidative Phosphorylation

PBS Phosphate Buffered Saline

PEO Progressive External Ophthalmoplegia RCR Rolling-Circle replication

RDR Recombination-dependent replication RNA Ribonucleic Acid

RITOLS RNA Introduced Throughout the Lagging-Strand ROS Reactive oxygen species

SDSA Synthesis-dependent strand annealing SDM Strand-Displacement model

SIMH Stress-Induced mitochondrial hyperfusion SSA Single-strand annealing

SSB Single-strand Binding protein ssDNA single-stranded DNA

SSBR Single-strand break repair SSC Saline-sodium citrate buffer

TIM Translocase of the Inner Membrane TOM Translocase of the Outer Membrane TAS Termination-associating sequence TLS Translesion

UV Ultraviolet light UVB Ultraviolet B light

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LIST OF ORIGINAL PUBLICATIONS

This thesis is based on data presented in the following articles, referrred to by the roman numbers I-III.

I Torregrosa-Muñumer R, Goffart S, Haikonen J, and Pohjoismäki J. (2015). Low doses of UV and oxidative damage induce dramatic accumulation of mitochondrial DNA replication intermediates, fork regression and replication initiation shift. Mol Biol Cell. 2015 Nov 15;26(23):4197-208. doi:

10.1091/mbc.E15-06-0390. Epub 2015 Sep 23.

II Torregrosa-Muñumer R, Forslund J, Goffart S, Stojkovic G, Pfeiffer A, Carvalho G, Blanco L, Wanrooij S and Pohjoismäki J. Primpol is required for replication re-initiation after mitochondrial DNA damage. PNAS. 2017 Oct.

24;114(43):11398-11403. doi:10.1073/pnas.1705367114.

III Torregrosa-Muñumer R, Blei D, Zsurka G, Goffart S, Kunz W.S and Pohjoismäki J. Replication fork rescue in mitochondria (Manuscript).

The original publication have been reproduced with the permission of American Society for Cell Biology (I) and National Academy of Sciences of United States of America (II).

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AUTHOR’S CONTRIBUTION

I) The author participated in the development of the idea, planing of the experiments and writing of the paper together with his supervisors.

Experiments for the supplementary figure 3 were conducted by Haikonen under the supervision of the author, while the author performed all the other experiments.

II) The author was responsible of developing the idea, planning the experiments and writing the paper together with his supervisors and collaborators. The author carried out the majority of cell culture experiments. Generation of Primpol inducible cell lines, in vitro experiments (Figure 2, S3 and S5) and cellular fractionation and co-inmunoprecipitation (Figure S7) were conducted at Prof. Wanrooij´s laboratory (Umeå University). Additional in vitro experiments (Figure S4) and generation of primary +/+ and -/- Primpol MEF cells were performed at Prof. Blanco´s group (Autonoma University of Madrid). Joint first authorship.

III) The author developed the idea, planned the experiments and wrote the manuscript together with his supervisors and collaborators. The HEK239 MGME1-KO cell line was generated at Prof. Kunz´s laboratory (University of Bonn, Germany), the MGME1-wt construct for transfection was generated at Dr./Prof. Dziembowsky´s laboratory (University of Warsaw, Poland) and Flp- In T-Rex 293 TWNK wild-type and LD mutant cell lines were generated by the group of Dr. Spelbrink (University of Tampere, Finland). The author performed all the experiments.

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1 INTRODUCTION

1.1 DISCOVERY OF MITOCHONDRIA

The term “mitochondrion” was originally coined by Carl Brenda in 1898, who derived it from the Greek words mito (thread) and condrion (granule) (Brenda, 1898).

Due to their appearance in early microscopic studies, mitochondria were described in the first half of the 19th century as “granules” (Kölliker, 1853). In 1890, the German pathologist and histologist Richard Altmann concluded that these structures, resembling bacteria, were living beings inside cells and performing vital functions (Altman, 1890). Some years later, in 1913, the German Otto Heinrich Warburg discovered that these “grana,” as he defined mitochondria, could consume oxygen (Warburg, 1913), although the principles of the respiratory chain would not start to be understood until the discovery of cytochromes in 1925 (Keilin, 1925). However, deeper biochemical studies of mitochondria were limited by technical issues, which were finally overcome by the Belgian cell biologist Albert Claude, who in the 1940s designed a differential centrifugation method to isolate functional mitochondria (Claude, 1946). This improvement finally led to the discovery in the 1960s of a small piece of DNA inside mitochondria, the mitochondrial DNA (mtDNA), by Margit and Sylvan Nass in Sweden (Nass & Nass, 1963) and Hans Tuppy’s group in Austria (Schatz et al., 1964). The mtDNA encodes several factors essential for cellular energy production and the machinery needed for their translation. Thus, in recent years, mtDNA maintenance has gained attention, as mutations or damaged mtDNA have been associated with a number of diseases, aging and cancer (Ernster et al., 1959; Holt et al., 1988; Gorman et al., 2016).

1.1.1 Origin of mitochondria

Different theories have been developed to explain the origin of mitochondria, most of which recognize an early symbiotic relationship as a key feature of the process. The best known is perhaps that of Lynn Margulis (formerly Sagan), who in 1967 proposed the serial endosymbiosis theory to explain the origin of mitochondria and other organelles (Sagan, 1967). According to this theory, mitochondria were originally aerobic -proteobacteria which were engulfed by a primitive anaerobic eukaryotic cell. Adding a new ecological context, William Martin and Miklós Müller introduced the hydrogen hypothesis in 1998, which proposed the fusion of a hydrogen- dependent archeabacterium and a hydrogen-producing -proteobacterium (Martin and Muller, 1998). In their view, hydrogen rather than oxygen boosted the symbiosis, and the host was another prokaryote. One of the latest theories, proposed by David Baum and Buzz Baum in 2014, is the inside-out theory, according to which an -

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proteobacterium (future mitochondrion) initially lived on the surface of an archeabacterium (future nucleus). It later started to form protrusions or “blebs”

around the free-living -proteobacterium to increase the contact surface for material exchange. Eventually, the enlarged protrusions enclosed the proto-mitochondrion and constituted the plasma membrane, with the space between blebs converted into the endoplasmic reticulum (Baum and Baum, 2014).

1.2 MITOCHONDRIA STRUCTURE AND FUNCTIONS

1.2.1 Mitochondrial membrane and dynamics

Mitochondria are enclosed by two phospholipid bilayer membranes, the outer and inner membranes, which constitute different sub-compartments: the intermembrane space (between membranes) and the matrix (enclosed by the inner membrane) (Figure 1). The inner and outer membranes differ in permeability. The outer membrane is rich in the protein porin, which forms channels that allow the free passage of molecules of 5 kDa or less into the intermembrane space. Therefore, the ionic composition and pH of the intermembrane space are the same as those of the cytoplasm, namely pH 7.4. In contrast, the inner membrane is less permeable, which permits the creation of an electrochemical gradient across the inner membrane, causing the matrix to have a different molecular composition and pH (pH 7.9). The inner membrane forms folds or invaginations called “cristae,” which increase the surface area for ATP production through oxidative phosphorylation and tend to be more abundant in tissues with high energy demand. MtDNA is anchored to the inner membrane, close to endoplasmic reticulum (ER)-mitochondrial junctions (Iborra et al., 2004, Rajala et al., 2014, Gerhold et al., 2015). About 99% of the mitochondrial proteins are synthesized in the cytosol and imported post-translationally into mitochondria (Calvo and Mootha, 2010). Unfolded mitochondrial proteins are normally directed into the appropriate mitochondrial compartment, primarily by signal sequences. The terminal sequence in the N-terminus is generally used for mitochondrial proteins localized in the matrix and is cleaved after import. In other cases, the signal is internal and not removed. Protein translocation through the outer and inner membranes is mediated by the translocation complexes TOM and TIM, respectively (Chacinska et al., 2009).

Mitochondria form a dynamic and complex network regulated by fusion and fission processes, which induce mitochondrial merging or fragmentation, respectively. A tight balance between both processes is crucial to keep pace with metabolic demands and guarantee the elimination of damaged organelles by mitophagy. Mitochondrial fusion allows the mixing of contents between mitochondria and has beneficial effects under conditions of high energy demand. It

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23 is mediated by three GTPases: the mitofusins MFN1 and MFN2, and L-OPA1. While mitofusins are involved in outer membrane fusion, OPA1 is needed for inner membrane fusion. In contrast, mitochondrial fission is involved in mitochondrial division, mitochondrial transport, apoptosis and removal of damaged mitochondria through mitophagy. It is mediated by the accumulation of S-OPA1 and obtained from the cleavage of L-OPA1, as well as by DRP1, which forms a ring structure at the site of division (Wai and Langer, 2016). The importance of mitochondrial fusion for mtDNA maintenance is highlighted by a double-knockout mouse model of MFN1 and MFN2, as the absence of both mitofusins results in mtDNA instability (Chen et al., 2010). Similar effects were observed in human mtDNA with mutant OPA1 (Milone et al., 2009). Mitochondrial fusion has been linked to adaptation mechanisms responding to certain types of stress. This phenomenon is known as stress-induced mitochondrial hyperfusion (SIMH) and is characterized by enlarged or hyperfused mitochondria as a response to modest levels of toxic agents such as UVC light or actinomycin D (an inhibitor of mitochondrial protein synthesis). The mixing of contents between mitochondria seems to improve their resilience, as the induction of SIMH in cells enhances ATP synthesis and mitigates cellular stress, based on delayed apoptotic activation. This response is dependent on MFN1 and OPA1, as mutations in these genes increases their sensitivity to stress (Tondera et al., 2009).

Figure 1. Mitochondrial structure. The mitochondrion is surrounded by two phospholipid membranes, the outer and inner membranes, creating an intermembrane space (pH 7.4). The inner space is a denominated matrix and has a different ionic composition (pH 7.9). The inner membrane forms invaginations known as cristae, in which the oxidative phosphorylation reactions take place. The mtDNA is organized in nucleoids anchored to the inner membrane.

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Damaged mitochondria are targeted to mitophagy, which degrades them as part of the cellular quality-control process. During mitophagy, the mitochondrion is initially enclosed by a double-membrane structure, the autophagosome, which fuses with endolysosomes to form an autolysosome, in which the mitochondrion is finally degraded by lysosomal hydrolases (Hamacher-Brady and Brady, 2016). Mitophagy is mediated by the kinase PINK1 and the E3 ubiquitin ligase Parkin, which have been found to be mutated in some cases of familiar Parkinson’s disease, thereby linking this disease with mitochondrial dysfunction (Kitada et al., 1998, Valente et al., 2004).

Interestingly, Parkin may interact with mtDNA and protect it against oxidative stress, as overexpression of Parkin in human cells results in increased mtDNA and mtRNA levels, and improve s recovery after H2O2 exposure. In contrast, fibroblast- lacking Parkin accumulates more oxidative damage and shows reduced mtDNA levels (Rothfuss et al., 2009). Parkin may endorse mtDNA integrity while inducing the elimination of the more damaged mitochondria in order to stimulate cell survival.

1.2.2 Mitochondrial functions

Mitochondria are essential for most eukaryotic cells as they are the main source of cellular energy. However, mitochondria have also other essential functions in cellular biogenesis, such as regulation of apoptosis, biosynthesis of Fe-S clusters and steroids, maintenance of calcium homeostasis and heat production.

The most important mitochondrial function is energy production through the oxidation of food-derived molecules in a process termed “oxidative phosphorylation” (OXPHOS) (Figure 2), a term coined by the Spanish Nobel laureate Severo Ochoa while studying pyruvate oxidation in the brain (Ochoa, 1940). In this metabolic pathway, the acetyl-CoA derived from sugars (glycolysis) or fatty acids (- oxidation) is fed into the Krebs or tricarboxylic acid cycle, where it is converted into CO², while the resulting energy is stored in the form of the reduced coenzymes NADH and FADH². These coenzymes are subsequently re-oxidized by the electron transport chain (ETC), which conveys the transfer of electrons from NADH and FADH² to the final acceptor oxygen. The transfer of electrons through the ETC is coupled to the pumping of protons into the intermembrane space, creating an electrochemical proton gradient between the mitochondrial membranes. This gradient drives the synthesis of ATP by ATP synthase, the fifth member of the OXHPOS complexes, in a process known as “chemiosmosis coupling” (Mitchell, 1961). Peter D. Mitchell was awarded the Nobel Prize in 1978 for the discovery of chemiosmotic coupling, while the discovery of the mechanisms underlying ATP synthesis (Boyer et al., 1973, Walker, 1994) was also worthy of a Nobel Prize, awarded to Paul D. Boyer and John E. Walker in 1997.

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25 Figure 2. Oxidative phosphorylation and mitochondrial ATP production. The ETC consists of four complexes anchored to the inner mitochondrial membrane and coupled to ATP synthase.

Initially, the electrons from NADH are transferred to Complex I (NADH dehydrogenase) and the electrons from FADH2 are transferred to Complex II (succinate dehydrogenase).

Complexes I and II then pass the electrons to ubiquinone or coenzyme Q, which in turn transfers them to Complex III (cytochrome b-c). Finally, Complex III passes the electrons to reach Complex IV (cytochrome C oxidase), which transfers them to molecular oxygen and generates two molecules of water. The transfer of electrons across the ETC complexes is coupled to the pumping of protons into the intermembrane space by complexes I, III and IV.

This creates a proton gradient that is used by ATP synthase (or Complex V of the OXPHOS chain) to transform ADP + Pi into ATP, boosted by the favorable translocation of protons from the intermembrane space into the matrix. Electron leakage, which drives the production of reactive oxygen species, mainly occurs at Complexes I and III.

Under normal conditions, Complex IV of the ETC transfers electrons to molecular oxygen (O²), which is reduced, resulting in the generation of two molecules of H²O.

However, electrons can exit the ETC before reaching Complex IV, mainly at Complexed I and III, and incompletely reduce O² to form the radical superoxide ·O^2- , which in turn can be transformed into other types of reactive oxygen species (ROS).

Due to the leakage of electrons from the ETC, mitochondria are the main source of endogenous ROS (Jastroch et al., 2010). At low levels, ROS are necessary for cellular signaling, but they can oxidize at high concentrations and damage macromolecules

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such as DNA, proteins and lipids, in a condition known as “oxidative stress”

(Schieber and Chandel, 2014). Due to its close proximity to the ROS generation sites in mitochondria, mtDNA is thought to be especially prone to oxidative damage.

Although only 1% of the mitochondrial proteins are mitochondrially encoded, they constitute core components for the OXPHOS chain. As a result, the accumulation of mutant mtDNA is not only linked to mitochondrial disorders, characterized by an impaired mitochondrial ATP production, but also to cancer and aging (Taylor and Turnbull, 2005).

Another significant function mediated by mitochondria is programmed cell death or apoptosis. This mechanism was initially linked with mitochondria in 1996 (Zamzami et al., 1996) and plays a key role during normal development in the elimination of unwanted cells. Apoptosis is also essential for the removal of infected, damaged or malignant cells. The mitochondrial or intrinsic pathway of apoptosis is initially mediated by Bcl-2 family proteins, which are divided into three subgroups:

pro-apoptotic Bax-like proteins, pro-apoptotic BH3 proteins and anti-apoptotic Bcl2- like proteins. Upon receiving an apoptotic stimulus, such as extensive DNA damage, the pro-apoptotic protein Bax translocates into mitochondria and interacts with a protein named Bak to form the apoptotic pore, while the anti-apoptotic Bcl2-like proteins are inhibited by BH3 proteins. This triggers the permeabilization of the mitochondrial membrane, which in turn provokes the release of Cyt c into the cytoplasm. The cytoplasmic Cyt c forms a complex with Apaf-1 and pre-Caspase-9, the so-called apoptosome, which finally activates the executioner Caspase-3, leading to the indiscriminate degradation of proteins. Additional pro-apoptotic proteins are released from the mitochondria to the cytosol and facilitate the activation of caspases such as Smac and HtrpA2, which prevent inhibitors of apoptosis factors such as XIAP from blocking caspase activation, as well as AIF (apoptosis induction factor), which is translocated into the nucleus and leads to DNA degradation (Wang and Youle, 2009).

1.2.3 Mitochondrial nucleoids

It was long assumed that mtDNA was naked and therefore likely more susceptible to DNA damage. However, it appeared that mtDNA forms a complex with proteins to constitute a packed-structure-denominated nucleoid for its analogy to bacterial DNA nucleoids (Spelbrink, 2010). Nucleoids have been proposed to be the mitochondrial unit of inheritance (Jacobs et al., 2000) and may serve to protect mtDNA from damage and bring together several copies of mtDNA in order to facilitate DNA repair (Holt et al., 2007).

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27 The number of mtDNA molecules per nucleoid is still under debate and depends on the resolution of the optical system used. The lowest estimated number is 1.4 mtDNA molecules per nucleoid in different mammalian cells (Kukat et al., 2011).

Among the proteins associated with mtDNA, the most abundant is TFAM, a protein involved in mtDNA maintenance and transcription. In analogy to nuclear histones, bacterial HIF and HU proteins, TFAM coats and protects mtDNA, forming the structural basis for the nucleoid (Kanki et al., 2004). In mammals, TFAM possesses a non-sequence-specific DNA-binding capacity (Fisher and Clayton, 1988) and its levels correlate linearly with mtDNA levels, as TFAM knockout mice lose their mtDNA (Larsson et al., 1998). Conversely, depletion of mtDNA with ethidium bromide (EtBr) in human cells provokes the loss of TFAM protein (Seidel-Rogol and Shadel, 2002), further confirming their interdependency. In addition, overexpression of TFAM in human cells results in a significant depletion of mtDNA and RNA transcripts, apparently caused by the stalling of the replisome due to an increasingly packaged template (Pohjoismaki et al., 2006). Other mtDNA maintenance proteins identified in the nucleoid include the helicase TWNK, the DNA polymerase Pol  and the single-stranded binding protein mtSSB (Garrido et al., 2003). Co-localization studies of mtDNA with two key components of the replisome, TWNK and mtSSB, reveal that only a subset of nucleoids is engaged in mtDNA replication at any given time (Rajala et al., 2014).

It has been proposed that nucleoids have a layered structure (Bogenhagen et al., 2008) with a core, in which mtDNA replication and transcription takes place, and a peripheral area, in which the polycistronic RNAs are processed and translated.

Intriguingly, the newly synthesized mtRNA remains close to the nucleoids and is stored in mitochondrial RNA granules for up to 40 min (Iborra et al., 2004). These RNA granules constitute discrete sub-compartments thought to be assembled around the newly transcribed mtRNA transcripts and in close vicinity to transcriptionally active nucleoids. In these granules, primary mitochondrial transcripts are processed and matured before being assembled into mitoribosomes for subsequent translation and insertion into the inner membrane (Jourdain et al., 2016). Nucleoids are attached to the mitochondrial inner membrane, as several mtDNA maintenance proteins co-purify with proteins localized in the inner membrane, such as VDAC (Bogenhagen et al., 2003). Around 35% of nucleoids have a less symmetric contour than the normal ellipsoidal shape, suggesting the inner membrane might influence the nucleoid shape (Brown et al., 2011). Nucleoids localize close to the cytoplasmic translation machinery at ER junctions, which are also involved in lipid homeostasis. This co-localization may facilitate the coordination between cytoplasmic and mitochondrial protein synthesis, and the assembly of OXPHOS complexes onto the inner membrane, as well as link membrane growth and mtDNA replication during mitochondrial division (Iborra et al., 2004, Rajala et al., 2014, Gerhold et al., 2015, Lewis et al., 2016).

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1.3 HUMAN mtDNA

The human mtDNA is a 16,659 bp circular, double-stranded DNA molecule (dsDNA). Human mtDNA was the first portion of the human genome to be fully sequenced, in 1981 (Anderson et al., 1981). The mouse mtDNA genome, published some months later, is only marginally smaller (16,303 bp) and very similar in sequence and organization (Bibb et al., 1981). In animals, mitochondrial DNA can adopt different topological forms. Along with circular monomers (supercoiled, closed or open circular forms), mtDNA can form catenanes with two or more interconnected genomes and concatenated head-to-tail circular dimers (Pohjoismaki and Goffart, 2011). In contrast to the diploid nuclear genome, mtDNA exists in several hundreds to thousands of copies per cell (Satoh and Kuroiwa, 1991). Despite the higher gene copy number, mtDNA represents only around 1% of the total cellular DNA. It contains 37 genes encoding 13 core proteins of the ETC, together with 22 tRNAs and two rRNAs required for mitochondrial protein synthesis (Figure 3). The two strands of the mammalian mtDNA double-helix have differing G:C content, enabling their separation as heavy (H) and light (L) strands using CsCl-density gradient centrifugation. MtDNA is compact: it does not contain introns and the coding genes are densely organized. However, mtDNA has two non-coding regions:

a 30-nucleotide sequence harboring the replication origin for the L-strand (O^L) and a 1.1 kb sequence known as the major non-coding region (NCR), which contains the transcription promoters for the light strand promoter (LSP) and the heavy strand promoter (HSP), as well as the replication origin of the H-strand (OH) and other regulatory elements (Figure 3).

In the NCR, mtDNA replication initiated at O^H can prematurely terminate at the termination-associated sequence (TAS), generating a 650-nt long DNA molecule called 7S DNA based on its sedimentation on a CsCl gradient. Much of the 7S DNA is hybridized to the template DNA, forming a triple-stranded structure in the NCR region called a displacement loop (D-loop) (Arnberg et al., 1971). The frequency of mtDNA molecules containing a D-loop/7S DNA varies from 10% to 90%, depending on the cell line, tissue or species (Nicholls and Minczuk, 2014). The function of the 7S DNA remains unclear, although the expensive energetic cost for its maintenance and turnover indicates it is essential. Several roles associated with mtDNA replication have been suggested, including replication initiation, termination and recombination (Nicholls and Minczuk, 2014). The 3’ terminus of the 7S DNA can be extended during replication, acting as a primer for the leading-strand synthesis, while the triple strand may serve as a fork barrier to facilitate the encounter of the two replication forks in the last step of replication. The detection of high levels of cruciform molecules in the NCR region and abundant breakpoint sites downstream the 3´ end of the D-loop suggests the 7S DNA might be involved in recombination. The more open structure of the D-loop may facilitate strand invasion events.

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29 Figure 3. Schematic diagram of the human mtDNA genome and the major NCR. The human mtDNA encodes 13 proteins, 22 tRNAs and 2 rRNAs. The NCR (zoomed area) includes the promoters for the LSP and HSP transcription, the origin of replication for the H-strand (OH), three conserved sequence blocks (CSB1-3) and the replication TAS. Unidirectional replication initiation has been mapped in the NCR region near the CytB gene (Ori-b) (Yasukawa et al., 2005). Bidirectional replication of mtDNA can start in a wider region, spanning several kb downstream OH, termed as Ori-z (Bowmaker et al., 2003). The D-loop is a structure generated by replication initiated at OH and prematurely terminated at TAS, creating a short DNA fragment termed as 7S DNA. Modified from Uhler and Falkenberg, 2015.

The D-loop may be also be important for the recruitment of proteins involved in mitochondrial DNA maintenance, as the elimination of 7S DNA might expose the replication origin, facilitating replication initiation. In this regard, mtSSB seems to stabilize this structure, while TFAM resolves it in vitro (Takamatsu et al., 2002). On the other hand, the D-loop may have evolved as a protection mechanism against DNA breakage. It may act as a shield for the cis elements contained in the NCR and be necessary for mtDNA maintenance, which is supported by the absence of mtDNA

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deletion in this region (Nicholls and Minczuk, 2014). Experimentally, 7S levels are very sensitive to manipulation of mtDNA maintenance proteins, although MGME1 seems to be specifically involved in its turnover (Kornblum et al., 2013).

1.3.1 Inheritance of mtDNA

In mammals, mtDNA is exclusively maternally inherited, although some cases of paternal leakage have been reported (Schwartz and Vissing, 2002, Greiner et al., 2015). The lack of sexual recombination implies the entire molecule has the same genetic history, making mtDNA an effective tool in tracing maternal lineages and their evolutionary histories. All mtDNA within a cell are typically genetically identical, i.e., homoplasmic, due to the segregation bottleneck during germ line development (Floros et al., 2018). When pathological mutations in mtDNA occur, they are buffered by the wild-type genomes and maintained in heteroplasmy as a mixed population of wild-type and mutant mtDNA. The level of heteroplasmy can vary between cells of the same tissue, between organs of the same individual and between individuals of the same family. A randomly determined pattern of inheritance has been proposed to explain why mtDNA mutations do not affect all cells equally (Stewart and Chinnery, 2015), although heteroplasmy is more frequent in some tissues than in others. As there are hundreds of mtDNAs per cell, the pathogenicity of the mutation depends on the ratio of mutant to wild-type mtDNA.

The pathological mutation typically needs to exceed a threshold of around 60–80%

before the biochemical defect is detectable (Boulet et al., 1992), indicating that mitochondrial mutations are functionally recessive and can be rescued by inter- mitochondrial complementation (Nakada et al., 2001).

1.4 DNA REPLICATION

DNA replication is the process by which a DNA molecule is duplicated to form two identical copies. In the case of the replication of double-stranded or duplex DNA, the process is semiconservative, as each daughter molecule conserves one strand from the parental molecule (Meselson and Stahl, 1958). Due to the intertwined nature of the duplex molecule, the ATP-dependent activity of a helicase enzyme is required to separate the annealed DNA strands and expose single-stranded DNA as a template (Costa et al., 2013). Unwinding of the double-stranded DNA provokes negative supercoiling in the front of the opening replication fork, which is relieved by the activity of topoisomerases transiently cleaving the DNA to release the tension (Pommier et al., 2016). The exposed single-stranded DNA is temporally covered by single-stranded binding proteins (SSB), preventing the template from re-hybridizing and stabilizing it structurally (Antony and Lohman, 2018). DNA synthesis is

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31 mediated by DNA polymerases, which catalyze the addition of deoxyribonucleotides (dNTPs) to the 3´-OH end of the newly synthetized strand, establishing a 5´-to-3´

direction of synthesis (Johnson and O'Donnell, 2005). The DNA template determines which of the four dNTPs is added in a complementary fashion. However, DNA polymerases cannot initiate DNA replication by themselves and require an RNA primer synthetized by a dedicated primase enzyme (Costa et al., 2013). The replication fork can be depicted as a Y-form structure, in which the short arms represent the newly synthesized daughter strands and the stem represents the intertwined parental DNA (Figure 4). Due to the 5´-to-3´ nature of DNA synthesis, the replication fork is asymmetric, with the leading strands being synthetized continuously and the lagging strand discontinuously (Snedeker et al., 2017).

Compared with the synthesis of the leading strand, which requires a single priming event, lagging-strand synthesis is primed many times, creating 1–2 kb DNA fragments known as Okazaki fragments (Okazaki et al., 1967), which are ligated into a continuous DNA strand by a DNA ligase.

Figure 4. Schematic representation of a DNA replication fork. The direction of DNA synthesis is always 5´-to-3´, which provokes one strand to be synthesized continuously (leading strand) and the other discontinuously (lagging strand). The synchronous replication of both strands requires a tight coordination of the replication machinery.

1.5 mtDNA REPLICATION

It is generally accepted that mitochondria constantly replicate mtDNA, independently of the cell cycle (Clayton, 1982). Cells must replace the mtDNA lost due to the mitochondrial turnover or produce additional mtDNA when mitochondrial biogenesis is upregulated as a response to a physiological stimulus. In

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addition, mitotic cells need to replicate their mtDNA to maintain its copy number.

Due to the evolutionary history of mitochondria, the mtDNA replication machinery in eukaryotes is essentially prokaryotic and viral (phage-derived) in its constituents.

Intriguingly, the mechanisms of mtDNA replication have evolved differently in the different eukaryote lineages, representing almost all possible replication modes, including recombination-dependent replication, rolling-circle replication, theta- replication and strand-displacement mechanisms. To further complicate matters, more than one mechanism might co-exist in the same group of organisms. For instance, at least three different mtDNA replication mechanisms have been proposed to exist in mammals (see section 1.8.3). This diversity makes it difficult to understand how mtDNA is replicated in any given organism; as a result, there is currently an ongoing, energetic debate about how mtDNA is duplicated in each eukaryote kingdom. Further studies are needed to elucidate this complexity.

1.5.1 mtDNA replication in fungi and plants

Fungi and plants have developed—or retained—similar mechanisms for mtDNA replication, including rolling-circle or sigma () replication (RCR) and recombination-dependent replication (RDR).

With a low level of compaction, yeast mtDNA can span up to several dozen kilobases. It was long assumed that yeast mtDNA is circular, similarly to animal mitochondrial genomes. However, a combination of pulse-field gel electrophoresis and electron microscopy studies revealed that mtDNA in Saccharomyces cerevisiae and Candida glabrata consists mostly of multimeric linear forms with only a small portion of circular molecules, some of which have a tail (known as lariat structures) (Maleszka et al., 1991, Williamson, 2002). Driven by these observations, at least two strategies have been proposed for yeast mtDNA replication: RCR and RDR (Figures 5 and 6).

Circular molecules can be replicated through an RCR mechanism (Figure 5), supported by the observation in C. glabrata and S. cerevisiae of lariat structures and linear molecules longer than the genome-unit length (Maleszka et al., 1991). The tails of the lariat structures are mainly double-stranded, suggesting coupled-leading and lagging-strand synthesis. On the other hand, the linear mtDNA molecules found in C. albicans and C. parapsilosis have been suggested to be replicated through RDR, supported by observations using the two-dimensional agarose electrophoresis (2D- AGE) of abundant recombination intermediates and complex branched molecules that are partially single stranded (Gerhold et al., 2010, Gerhold et al., 2014).

According to this model, replication is initiated when a dsDNA molecule is invaded by an ssDNA stretch, after which DNA synthesis progresses either by coupled or

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33 uncoupled mechanisms (Figure 6). The detection of RNA:DNA hybrids at the transcription-promoter sites suggests that transcription may facilitate the strand invasion process. It has been suggested that the mtRNA polymerase may mediate the priming of DNA synthesis at specific sites rich in GC, known as rep/ori in species such as S. cerevisiae and its relatives. Although still controversial, the hypothesis is supported by the observation that the mtRNA polymerase can efficiently catalyze the priming of DNA synthesis on an ssDNA template in vitro (Ramachandran et al., 2016).

Figure 5. Rolling-circle replication or sigma () replication. Unidirectional and strand- asynchronous replication may be initiated by the nicking of one strand by an endonuclease or strand-invasion (not shown), which both provide a 3´-OH that can be extended by the DNA polymerase. The elongation of the leading strand on a circular template generates a lariat (- like) structure. Several rounds of replication create circularly permutated head-to-tail concatemers, which are multiples of the genome length. The concatemers are resolved to genomic monomers in a subsequent process, after which the displaced parental strand is synthesized, either by a single priming event or by forming Okazaki fragments (Gilbert and Dressler, 1968).

The most recently proposed replication model suggests that yeast mtDNA replication proceeds origin-independently through an unconventional mechanism that does not require RNA primers and combines RDR, RCR and template switch, explaining the co-existence of linear and circular mtDNA molecules in the same organelle. According to this model, the invasion of a circular genome by a linear dsDNA molecule with a 3´overhang (resected by nucleases) may initiate the synthesis of the leading strand (RDR), which can subsequently be replicated by the sustained elongation on the circular template, creating a lariat structure (RCR). The lagging-strand synthesis, in contrast, would be initiated by the DNA polymerase

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switch from the circular template to the recently synthesized single-stranded tail (Chen and Clark-Walker, 2018).

Figure 6. Recombination-dependent replication. This replication mode is initiated by the invasion of an ssDNA fragment, originated for instance by the resection of a double-strand break by a 5´-to-3´ exonuclease. The 3’ single-stranded overhang is covered by SSB proteins and can anneal by homology to another single-stranded stretch covered by SSB to form a branch structure or Holliday junction. The invading strand provides the 3’-OH required to initiate the DNA synthesis of the leading strand, while lagging-strand synthesis may proceed via primase and formation of Okazaki fragments. For more information, see section 1.7.1.

The mitochondrial DNA polymerase in S. cerevisiae is known as Mip1. Compared to the heterotrimeric mammalian DNA polymerase Pol , Mip1 is monomeric, suggesting a different regulation of its activity. At least two helicases have been found in yeast mitochondria: Pif1 and Hmi1p. Pif1 is likely required for yeast mtDNA repair and the recognition of recombinogenic signals (Lahaye et al., 1993).

On the other hand, although Hmi1p is essential for mtDNA metabolism, its helicase activity is not. Instead it has been suggested that, similarly to the E. coli PriA, it might be involved in replication restart by recruiting other proteins into the stalled replication fork (Monroe et al., 2005). Two recombination proteins have been

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35 identified in yeast: the Rad52-like Mgm101 (Zuo et al., 2007) and Rad51/RecA-like Mhr1 (Ling and Shibata, 2002). In canonical recombination, Rad52 recruits Rad51/RecA-like proteins to ssDNA to form nucleoprotein filaments, which initiate homology search along the template DNA, suggesting the yeast-homologous recombinases may facilitate strand invasion during RDR. Recombination junctions are resolved by a specific type of enzyme, namely a resolvase. Two resolvases have been identified in yeast mitochondria: YDC2 in Schizosaccharomyces pombe and CCE1 in Saccharomizes cereveisae (White and Lilley, 1996, Oram et al., 1998). Tetrameric SSB Rim1, homologous to E. coli SSB, has been identified as essential for mtDNA replication in yeast, as its deletion results in mtDNA depletion (Van Dyck et al., 1992).

The size of mtDNA in plants is vastly larger than in other organisms, varying from 200 kb to 11.3 Mb. Studies of mtDNA from Chenopodium album (L.) using electron microscopy revealed circular molecules with a partially single-stranded tail (lariat structure), as well as ssDNA linear molecules, suggesting that mtDNA in C.

album is replicated through RCR (Backert et al., 1996) (Figure 5). In another study using pulse-field electrophoresis, mtDNA in Arabidopsis taliana was found as branched linear forms larger than the size of the genome, with a small percentage of circular genomes (also known as master circles) and head-to-tail tandem repeats (Bendich, 1996). The fact that circular molecules in plant mitochondria are a minority, dwarfed by the abundant linear and branched linear molecules larger than the genome unit length (Bendich, 1996), has driven scholars to suggest that mtDNA replication in plants is dominated by recombination events, i.e., RDR (Oldenburg and Bendich, 2015) (Figure 6).

Two mtDNA polymerases have been identified in plants: PolIA and PolIB (also known as POPs). In sequence similarity they are closer to the bacterial PolI than to the animal mtDNA Pol  (Moriyama and Sato, 2014). A study of PolIA and PolIB mutants in A. thaliana indicates that both are needed for mtDNA replication, as the double mutant was lethal while the single mutants caused the reduction of the mtDNA copy number. In addition, PolIB seems to be important also for mtDNA repair, as PolIB mutants are more sensitive to DNA damage (Parent et al., 2011). DNA unwinding is catalyzed by the TWNK helicase, which in contrast to the animal TWNK has a functional primase domain and is able to prime DNA synthesis in vitro, suggesting it might also act as the mtDNA primase in plants (Diray-Arce et al., 2013).

Orthologues of RecA and Rad51 have been identified in A. thaliana mitochondria and have been suggested to be involved in recombination during DNA replication and repair (Khazi et al., 2003, Samach et al., 2011). In addition, two classes of single- stranded binding proteins have been described in plants, SSB and OSB, the presence of which is variable among photosynthetic eukaryotes. SSB seems to stimulate the homologous strand exchange of E. coli RecA, while OSB mutants have been shown

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to accumulate aberrant recombination products, indicating that both proteins may be important for mtDNA recombination (Edmondson et al., 2005, Zaegel et al., 2006).

1.5.2 mtDNA replication in invertebrate metazoans

At least two different mtDNA replication mechanisms have been proposed to exist in invertebrate metazoans: unidirectional strand-coupled or theta () replication in kinetoplastids, insects and echinoderms, and RCR in nematodes.

The kinetoplast DNA, the mitochondrial DNA in kinetoplastids such as Trypanosoma brucei, has an unusual and complex structure, forming a massive network of hundreds of catenated circular molecules resembling a medieval chain mail. There are two types of circles: maxicircles (20–40 kb, depending on the species), which encode the typical mitochondrial genes, and minicircles (0.5–10 kb, depending on the species), which encode guide RNAs required for editing the maxicircle transcripts. The former are present in a few dozen copies in the network, while the latter are present in their thousands (Lukes et al., 2002). Both minicircle and maxicircle molecules are duplicated through a unidirectional theta-like () or strand- coupled mechanism (Figure 7), mainly characterized by electron microscopy (Brack et al., 1972, Carpenter and Englund, 1995). Initially, minicircles are decatenated by a topoisomerase and become free minicircles, which are replicated by a strand-coupled mechanism. The progeny minicircles are gapped or nicked due to the discontinuous nature of DNA synthesis and the gaps are not immediately filled. In the last step, once all the minicircles are replicated, the gaps are refilled and the catenated network is restored with the help of a topoisomerase. Maxicircles follow a canonical strand- coupled replication. In contrast to mtDNA replication in other species, which occurs throughout the cell cycle, kDNA replication is restricted to the S-phase (Klingbeil et al., 2001).

The mtDNA in the fruit fly, Drosophila melanogaster, is a double-stranded circular molecule DNA of 19.52 kb in size (Lewis et al., 1995). The genome has a large A+T- rich NCR from which mtDNA duplication starts unidirectionally (Goddard and Wolstenholme, 1978). Interestingly, the two initial studies using electron microscopy were contradictory. One reported mostly duplex replication intermediates with only short ssDNA stretches, suggesting a strand-coupled replication mechanism (Rubenstein et al., 1977). In contrast, the second study found evidence of a highly asymmetric or strand-asymmetric replication mode, implicating long regions of ssDNA, as one strand was found to be almost fully replicated before the synthesis of the other strand had started (Goddard and Wolstenholme, 1978). Thirty years later, another study using 2D-AGE shed light on this controversy (Joers and Jacobs, 2013), reporting that the Drosophila mtDNA replication intermediates are mainly dsDNA,

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37 as they were resistant to S1 digestion, supporting a strand-coupled replication mechanism. However, short ssDNA regions are present nearby the rRNA gene region, supposedly caused by a delayed lagging-strand synthesis as a consequence of the controlled passage of transcription complexes meeting the replication fork from the opposite direction. Therefore two unidirectional theta-like () replication modes may co-exist in D. melanogaster: a predominant strand-coupled replication and a minor asynchronous replication mechanism (Figure 7).

Figure 7. Strand-coupled replication or theta () replication. Replication starts at specific replication origins in which the dsDNA is unwound, exposing ssDNA as a template for DNA synthesis and forming a replication bubble with one (unidirectional) or two (bidirectional) replication forks progressing throughout the DNA molecule and forming a -like structure. The synthesis of the leading strand proceeds continuously in the direction of the opening replication fork, while the lagging strand is synthesized in the opposite direction discontinuously, forming Okazaki fragments. Termination is believed to occur when the replication forks encounter each other at termination sites. The two daughter DNA molecules remain interlocked, forming hemicatenanes, which are subsequently resolved by a topoisomerase.

Similar partially and fully dsDNA replication intermediates have been reported from mitochondria of the sea urchin Strongylocentrotus purpuratus oocyte, suggesting a strand-coupled replication mechanism also occurs in echinoderms (Matsumoto et al., 1974). The double-stranded nature of these replication intermediates was later confirmed by 2D-AGE, with a prominent lagging-strand replication-initiation site recognized at two thirds of genome length downstream from the D-loop, which also seem to serve as a pause site for the leading-strand synthesis (Mayhook et al., 1992).

The mitochondrial genome of the nematode Caenorhabditis elegans is a circular dsDNA molecule of 13.8 kb containing two NCR regions (Okimoto et al., 1992), which by analogy with the mammalian mtDNA may serve as master regulators for

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the synthesis of the leading and lagging strands. However, although Y-form replication intermediates were observed using 2D-AGE, spanning fragments containing any of these NCR regions, replication bubbles were not detected, suggesting C. elegans mtDNA is replicated differently than in other known animals.

Analysis of C. elegans mtDNA by transmission microscopy reveals circular molecules with a long tail, also known as lariat (-like) structures. The absence of a bubble arc and the presence of lariat structures indicate that mtDNA replication in C. elegans follows an RCR mechanism (Figure 5), in which the leading and lagging strands are replicated synchronously, as no extensive ssDNA regions were detected. The enrichment of cruciform or recombination intermediates detected on the major NCR, which were partially sensitive to degradation by a resolvase, suggests the monomers may be resolved through recombination (Lewis et al., 2015). Although RCR seems to be a common mechanism for the replication of fungi and plant mtDNA, C. elegans is thus far the only metazoan in which this mechanism has been detected.

The mtDNA replication machinery present in invertebrates is very similar to that found in vertebrates, including mtSSB, Pol  and TWNK. However, slight differences exist, which may also explain the mechanistic differences observed in different metazoan taxa. For instance, compared to the mammalian heterotrimeric Pol  (formed by one  and two  subunits), Pol  in D. melanogaster is a heterodimer formed by one  and one  subunit (Wernette and Kaguni, 1986), while only a single

 subunit enzyme exists in C. elegans (Oliveira et al., 2015). In contrast, no Pol  homologs have been found in kinetoplastids, but they contain four distinct polymerases close to the bacterial DNA pol I (Pol IA, IB, IC and ID) which might have redundant or different roles during the replication of mini- and maxicircles (Klingbeil et al., 2002). In addition to these replicative polymerases, also two DNA repair polymerases are known from kinetoplastid mitochondria: Pol  and Pol  PAK (with a domain rich in proline, alanine and lysines), which are similar to the mammalian nuclear Pol  and likely involved in the gap-filling process of the daughter minicircles (Saxowsky et al., 2003), and one TLS DNA polymerase, TcPol, which is related to Pol  (Rajao et al., 2009). In addition, six different DNA helicases related to the yeast Pif1 helicase have been reported, although only three of them are essential (TbPIF1, TbPIF2 and TbPIF3) for mtDNA maintenance. TbPIF1 seem to be involved in the replication of minicircles, while TbPIF2 regulates the duplication of maxicircle DNA (Jensen and Englund, 2012).

1.5.3 mtDNA replication in mammals

Despite the advances made thus far, there is a lack of consensus regarding the mechanism by which mammalian mtDNA is replicated. Several different mtDNA replication mechanisms have been proposed: strand-displacement model (SDM)

Replication stress and damage tolerance in mammalian mitochondria

uef.fi

Dissertations in Forestry and Natural Sciences

RUBÉN TORREGROSA MUÑUMER

REPLICATION STRESS AND DAMAGE TOLERANCE IN MAMMALIAN MITOCHONDRIA

RUBÉN TORREGROSA MUÑUMER

REPLICATION STRESS AND DAMAGE TOLERANCE IN MAMMALIAN

MITOCHONDRIA

Rubén Torregrosa Muñumer

REPLICATION STRESS AND DAMAGE TOLERANCE IN MAMMALIAN

MITOCHONDRIA

ABSTRACT

ACKNOWLEDGEMENTS

LIST OF ABBREVIATIONS

LIST OF ORIGINAL PUBLICATIONS

AUTHOR’S CONTRIBUTION

CONTENTS

1 INTRODUCTION

1.1 DISCOVERY OF MITOCHONDRIA

1.2 MITOCHONDRIA STRUCTURE AND FUNCTIONS

1.3 HUMAN mtDNA

1.4 DNA REPLICATION

1.5 mtDNA REPLICATION