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DOCTORADO EN BIOCIENCIAS MOLECULARES

EXPLORING THE ROLE OF THE USP7 DEUBIQUITINASE IN CELL CYCLE CONTROL AND CANCER THERAPY

DOCTORAL THESIS

ANTONIO GALARRETA ABELLÁN

MADRID, 2020

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FACULTAD DE MEDICINA DEPARTAMENTO DE BIOQUÍMICA UNIVERSIDAD AUTÓNOMA DE MADRID

EXPLORING THE ROLE OF THE USP7 DEUBIQUITINASE IN CELL CYCLE CONTROL AND CANCER THERAPY

ANTONIO GALARRETA ABELLÁN

BSc, BIOCHEMISTRY

MSc, MOLECULAR BIOMEDICINE

The work presented in this Doctoral Thesis has been carried out at the Genomic Instability Group in the Spanish National Cancer Research Centre (CNIO, Madrid), under the supervision of Dr. Oscar Fernández- Capetillo Ruiz and Emilio Lecona Sagrado, and was funded by the Spanish Ministry of Economy and Competitiveness (FPI MINECO Fellowship).

Madrid, 2020

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A mi madre y a mi padre

A Belén

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“To live is to face one problem after another.

The way you face it makes a difference”.

Benjamin Franklin

.

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ACKNOWLEDGMENTS

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No puedo empezar a escribir estas líneas sin echar la vista atrás, y una vez más, darme cuenta de lo rápido que pasa el tiempo. Embarqué en esta etapa, experiencia y locura que puede llegar a ser hacer una tesis doctoral hace ya cuatro años. Cuatro años llenos de esfuerzo, trabajo, de grandes momentos, de muchas, muchas emociones, algunas mejores... y otras no tanto (pero eso es el doctorado, ¿no?), de momentos que te marcan. Al final el doctorado no es un viaje que haces solo, y por ello querría agradeceros a todos vosotros que de alguna u otra manera me habéis ayudado a crecer como científico, que me habéis apoyado, animado y acompañado en este trayecto.

En primer lugar, me gustaría dar las gracias a todo el laboratorio de Inestabilidad Genómica, tanto a los miembros presentes, como a los pasados. Más allá de todo lo que me hayáis enseñado, ha sido un placer trabajar con todos vosotros. Siempre se habla del laboratorio como una segunda casa (o la primera…), de que pasamos más horas juntos entre esas cuatro paredes que con cualquier otra persona… así que gracias por todos los buenos momentos en estos años.

Óscar, muchas gracias por haberme permitido trabajar en este espectacular proyecto bajo tu dirección. Lo he disfrutado mucho. Gracias por todas tus enseñanzas, por tener la puerta del despacho y el email siempre abiertos, y por inculcarnos que hay que tener siempre la big picture de lo que estamos haciendo, hacer experimentos sencillos, y cuando olisqueemos algo interesante… ir a por la one-million dollar question.

Emilio, “Master of Proteins”, gracias por todo. ¡No podría haber tenido un maestro mejor en esta etapa! Gracias por confiar siempre en mí y por toda la paciencia que has tenido conmigo, con mi “tranquilidad”. Me has formado, ayudado a mejorar en todos los aspectos del doctorado, frenado cada vez que descarrilaba y entraba en bucle... (¡puedes respirar tranquilo ya por ello!). Siempre me calaste cuando me pasaba algo, y estuviste disponible para darme consejos ante cualquier situación.

Mati, eres el soporte de este laboratorio, sin ninguna duda. ¡Es alucinante ver como siempre vas con una marcha más que el resto! Muchas gracias por estar ahí para darnos consejos ante cualquier problema que pudiera surgir. Vane, muchas gracias por toda la ayuda a lo largo de estos años, por todo el apoyo y toda la cantidad de inputs e interés mostrado acerca de esta tesis. Bárbara, gracias por todos los consejos, y la tranquilidad, y la calma que aportas al laboratorio.

A mis compañeros de travesía, antes de nada, os deseo todo lo mejor para lo que os queda de doctorado. ¡Espero que disfrutéis lo que os queda y no decaigáis! Sasha, compañera desde prácticamente mi primer día aquí. ¡Es un honor cederte el trono de

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para sembrar tu reino de terror y mantener a raya a todos los millennials/generación Z o lo que sea que tenemos ahora por el laboratorio (aunque sé que en el fondo no serás muy dura con ellos). Voy a echar de menos tu sentido del humor, ¡no lo dudes! Pablo, narrador de las historias más surrealistas que te puedas llegar a imaginar, gracias por hacer del laboratorio un lugar tan entretenido. Laura, la más organizada de todo el lab, y nuestra organizadora de eventos oficial. En tu honor el calendario de Outlook me dice que en estos minutos tengo que ponerme contigo. ¡No pierdas nunca todo tu positivismo! Elena, el pasillo es todo tuyo. Que será de mis días sin escuchar el dúo Elena-Pablo. Gracias por todas las veces que me habéis hecho reír, y por todo lo que he aprendido de vosotros de la cultura actual.

Marta, ¡gracias por estar dispuesta siempre a ayudar, y a generar buenos momentos en el lab! Sara, siempre queriéndonos echar una mano con cualquier cosa. Gracias por ello y por el buen rollo que transmites. Alicia, muchas gracias por todo ese trabajo que no es tan visible pero que nos hace la vida mucho más sencilla a todos.

A los antiguos miembros, empezando por el team DC, Sergio, que tu andadura por DC solo vaya a mejor. María, muchas gracias por tu ayuda cuando la necesité, and finally Tere, thank you for all the good moments, advice, chats during cultivos time…, I wish you all the best. Fede, ¡cuánta alegría y locura transmitías en el lab! Isa, gracias por todos los consejos que me diste cuando empecé. ¡Cuánta razón tenías! Cris, espero que te vaya muy bien por Viena, y ¿en una próxima nueva etapa? Y también a los que tuvieron un paso más breve por el lab, Patricia Ubieto; Patricia Cozar., Judith, espero que os vaya fenomenal, tanto en vuestras carreras como a nivel personal.

También querría agradecer a las Unidades del CNIO que han hecho posible, con su ayuda, que este proyecto haya salido adelante. Muchísimas gracias a los miembros de la Unidad de Microscopía Confocal. Diego, Manu, Jesús, Gadea… Bajar a la -2 es siempre sinónimo de echarse unas risas y pasar un buen rato. Gracias también a la Unidad de Citometría de Flujo, a Lola, Ultan, Miguel Ángel, Tania y Julia, por todo el apoyo recibido.

Gracias a la Unidad de Proteómica, a Javier y Eduardo, por su colaboración y ayuda cuando lo necesitamos.

Por supuesto gracias también al resto de grupos, a todas las personas que he podido conocer durante estos años y que han hecho más agradable mi estancia aquí, a cada persona que, con su ayuda, desde compartiendo el más sencillo de los protocolos, prestando reactivos, o compartiendo ideas, me han ayudado con este trabajo, y en general a las personas que hacen posible que sea increíble trabajar en el CNIO.

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No me puedo olvidad tampoco de vosotros, que durante estos años habéis estado preocupándoos por mí, animándome y, sobre todo, generando grandes recuerdos.

Alfonso, coincidir en Madrid ha sido una gran oportunidad de retomar esta amistad que, por cosas de la vida, se dejó un poco de lado. Clara y Rubén, otras dos personas de las que, tras taaaaantos años, no me puedo librar jajaja. Gracias por todos los buenos momentos que llevamos. Laura, Pablo, gracias también por todo vuestro apoyo durante todo este tiempo. Pablo, ¡eres el siguiente!

Y no podría dejarme a mi familia, que siempre han estado ahí en los buenos y en los malos momentos. Mamá, Papá, gracias por todo el apoyo que me habéis dado, por todo vuestro cariño y amor. Por haberme inculcado valores como el esfuerzo, el trabajo y la superación, que han hecho posible que ahora esté donde estoy. Siempre habéis luchado por mí. Gracias a mis yayos por todo su cariño y dedicación. Y por supuesto, gracias a mi tío Javier, mi hermano mayor, por todos tus consejos, tu implicación, y por todos los buenos momentos. ¡Todo el mundo debería tener un tío como tú! Siempre has sido un ejemplo para mí, un espejo en el que mirarme.

Y, por último, gracias a ti, Belén, por TODO. Por estar a mi lado todo este tiempo, apoyándome, compartiendo todas mis alegrías y aguantando todas mis desesperaciones.

Por cuidarme y animarme siempre que lo he necesitado, porque a veces no fue fácil. Por luchar siempre por sacar la mejor versión de mí mismo, por confiar ciegamente en mí y en mis posibilidades. Por implicarte en esta tesis. Por hacer que este camino no lo haya recorrido solo. Porque somos un equipo y porque esta tesis también es tuya. Gracias por todo el amor que me has demostrado. Te quiero.

Gracias.

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RESUMEN

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El modelo clásico del ciclo celular consiste en una secuencia de eventos ordenados que conducen a la duplicación del ADN y finalmente a la división celular. Para prevenir la inestabilidad genómica y muerte celular, la replicación del ADN y la división celular necesitan estar correctamente coordinadas. Sin embargo, cómo las células perciben que la replicación del ADN está terminada y cómo y cuándo se desencadena la mitosis aún no se comprende completamente. Durante los últimos años, muchos artículos han demostrado que modificaciones postraduccionales como la ubiquitinación, pueden regular todos estos procesos. En este contexto, recientemente describimos que la inhibición de la deubiquitinasa USP7 conduce a una terminación prematura de la replicación del ADN y a daño en el ADN por razones que desconocíamos. Durante esta tesis, hemos usado esta herramienta para investigar cómo o sí la terminación de la replicación del ADN es la señal que induce la entrada mitótica, asegurando que la segregación cromosómica no se inicie antes de que se haya replicado todo el genoma. Nuestros resultados sugieren que la inhibición de USP7 conduce a la ubiquitinación de varios factores de replicación y su desplazamiento de los replisomas, mientras que al mismo tiempo desencadena una activación prematura de las quinasas mitóticas (por ejemplo, CDK1) a lo largo del ciclo celular. Mecanísticamente, encontramos que los inhibidores de USP7 imitan los efectos de la inhibición de la fosfatasa PP2A. Todo ello da como resultado la condensación cromosómica prematura y la muerte celular, la cual no está mediada por P53. Por el contrario, la toxicidad de los inhibidores de USP7 puede contrarrestarse con los inhibidores de CDK1 o activadores de PP2A, que limitan la activación del programa mitótico. Nuestros resultados indican que los efectos anticancerígenos de los inhibidores de USP7 no dependen únicamente como se pensaba de P53, sino que están relacionados con la activación prematura de la maquinaria mitótica en las células que no han terminado la replicación del ADN. Además de su interés terapéutico frente al cáncer, nuestro trabajo sugiere la existencia de un código de señalización basado en ubiquitinación que coordina la terminación de la replicación del ADN con la entrada mitótica.

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ABSTRACT

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The classical model of the cell cycle consists of a sequence of ordered events leading to the duplication of DNA and ultimately cell division. To prevent genomic instability and cell death, DNA replication and mitosis need to be properly coordinated. However, how cells sense that DNA replication is finished, and how and when mitosis is triggered are yet not fully understood. During the last years, many papers have reported that post- translational modifications such as ubiquitination, can regulate all these processes. In addition, we recently described that the inhibition of the USP7 deubiquitinase, leads to a premature termination of DNA replication and DNA damage for reasons that remained unknown. During this thesis, we have used this tool to investigate how or if DNA replication termination is the actual signal for mitotic entry, ensuring that chromosome segregation is not initiated before the entire genome has been replicated. Our results suggest that USP7 inhibition leads to the ubiquitination of several replication factors and their displacement from replisomes, while at the same time triggers a premature activation of the mitotic kinases (e.g. CDK1) throughout the cell cycle. Mechanistically, we found that USP7 inhibitors phenocopy the effects of the phosphatase PP2A inhibition. This results in premature chromosome condensation and cell death, which is not mediated by P53. In contrast, the toxicity of USP7 inhibitors can be counteracted by CDK1 inhibitors or PP2A activators, both of which limit the activation of the mitotic program. Our results indicate that the anticancer effects of USP7 inhibitors are not solely dependent on P53 as previously thought, but also related to a premature activation of the mitotic machinery in cells that have not terminated DNA replication. Besides its interest for cancer therapy, our work suggests the existence of a ubiquitin-based signaling code that coordinates DNA replication termination and mitotic entry.

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INDEX

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ACKNOWLEDGMENTS ... 11

RESUMEN ... 17

ABSTRACT ... 21

INDEX ... 25

ABBREVIATIONS ... 31

INTRODUCTION ... 39

The cell cycle ... 41

1.1. Cell cycle regulators: CDKs and cyclins ... 41

1.2. Moving forward through the cell cycle: The Threshold model ... 42

DNA replication ... 43

2.1. DNA replication initiation ... 44

2.2. DNA replication elongation ... 44

2.3. DNA replication termination ... 45

2.4. Problems during DNA replication: Replication stress ... 47

Mitosis ... 48

3.1. The mitotic trigger is an irreversible cellular switch ... 48

3.2. From mitotic entry to anaphase ... 50

3.3. The APC/C complex and mitotic exit ... 50

Are DNA replication and mitosis really independent?... 51

4.1. DNA replication during mitosis (MiDAS) ... 51

4.2. The S/M checkpoint and premature mitotic activation ... 52

4.3. Coordinating DNA replication and mitosis ... 52

Ubiquitination and SUMOylation: and overview ... 53

5.1. The ubiquitin-proteasome signaling pathway... 53

5.2. The SUMOylation pathway ... 55

The deubiquitinase USP7 ... 56

6.1. USP7 and the regulation of the MDM2/P53 pathway ... 56

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6.2. USP7 and DNA replication ... 57

6.3. USP7 and mitosis ... 58

OBJECTIVES ... 59

MATERIALS AND METHODS ... 63

Cellular biology: ... 65

1.1. Cell lines ... 65

1.2. Treatments ... 65

1.3. Cell synchronization ... 65

1.4. Plasmids and transfections ... 66

1.5. RNA interference ... 66

1.6. High-Throughput Microscopy ... 66

1.7. Immunofluorescence ... 67

1.8. Live cell imaging ... 67

1.9. Flow cytometry ... 67

1.10. Metaphase spreads ... 68

1.11. Cell viability assays ... 68

Molecular biology and biochemistry ... 69

2.1. Protein extraction, Cell fractionation and Western blot ... 69

2.2. Immunoprecipitation with Ni-NTA agarose ... 70

2.3. Immunoprecipitation with Protein G Dynabeads ... 70

2.4. Isolation of proteins on nascent DNA (iPOND) ... 70

2.5. Liquid chromatography and mass spectrometry (LC-MS/MS) ... 71

2.6. Phosphatase assays ... 73

Data analysis ... 73

RESULTS ... 75

PART I: USP7 INHIBITON AS A MODEL FOR DNA REPLICATION TERMINATION .. 77

USP7 inhibition arrests cells in S-phase ... 79 USP7 inhibition recapitulates some of the hallmarks of DNA replication termination80

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USP7 inhibition induces disassembly of the replisome ... 82

PART II: DISSECTING THE ROLE OF USP7 ON MITOTIC ENTRY ... 85

USP7 inhibition triggers a premature activation of mitotic kinases ... 87

USP7 suppresses cyclin B/CDK1 activity throughout the cell cycle ... 91

USP7 interacts with and regulates the activity of PP2A ... 96

Suppression of PP2A activity by USP7 inhibitors drives activation of the mitotic program ... 99

USP7 inhibition induces mitotic problems that impairs cell division ... 100

PART III: A NOVEL MECHANISM OF ACTION FOR USP7 INHIBITORS AS ANTICANCER AGENTS ... 103

USP7 inhibition triggers mitotic catastrophe ... 105

Limiting CDK1 activity suppresses the toxicity of USP7 inhibitors ... 106

DISCUSSION ... 111

USP7 inhibition as a model for DNA replication termination ... 115

Dissecting the role of USP7 on mitosis ... 116

A novel mechanism of action for USP7 inhibitors ... 119

CONCLUSIONES ... 123

PARTE I: LA INHIBICIÓN DE USP7 COMO MODELO DE TERMINACIÓN DE LA REPLICACIÓN DEL ADN: ... 125

PARTE II: DISECCIONANDO EL PAPEL DE USP7 EN LA ACTIVACIÓN MITÓTICA: 125 PARTE III: UN NUEVO MECANISMO DE ACCIÓN PARA LOS INHIBIDORES DE USP7 COMO AGENTES ANTICANCERÍGENOS: ... 125

CONCLUSIONS ... 127

PART I: USP7 INHIBITION AS A MODEL FOR DNA REPLICATION TERMINATION: 129 PART II: DISSECTING THE ROLE OF USP7 ON MITOTIC ENTRY: ... 129

PART III: A NOVEL MECHANISM OF ACTION FOR USP7 INHIBITORS AS ANTICANCER AGENTS: ... 129

BIBLIOGRAPHY ... 131

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ABBREVIATIONS

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53BP1 Tumor suppressor p53 Binding Protein 1

Abs Absorbance

AKT Protein kinase B (PKB) ALL Acute lymphoblastic leukemia

APC/C Anaphase Promoting Complex/Cyclosome

APH Aphidicolin

ATCC American Type Culture Collection

ATP Adenosine Tri-Phosphate

ATR Ataxia Telangiectasia and Rad3 related ATRIP ATR Interacting Protein

AURKA Aurora A AURKB Aurora B

BSA Bovine serum albumin

BUB3 Budding uninhibited by benzimidazoles 3 homolog CAA Chloroacetamide

CCNB1 Cyclin B1

CCNB1 (S126P) Phosphorylated form of cyclin B1 at serine 126 CDC2 Cell division control protein 2

CDC6 Cell Division Cycle 6 CDC20 Cell Division Cycle 20 CDC25 Cell Division Cycle 25 CDC25A Cell Division Cycle 25A CDC45 Cell Division Cycle protein 45 CDH1 Cadherin 1

CDK Cyclin-Dependent Kinase

CDK1 Cyclin-Dependent Kinase 1

CDK1Y15P Phosphorylated form of CDK1 at tyrosine 15 CDK2 Cyclin-Dependent Kinase 2

CDK4 Cyclin-Dependent Kinase 4 CDK6 Cyclin-Dependent Kinase 6 CDK7 Cyclin-Dependent Kinase 7 CDT1 CDC10-depenent transcript 1

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CENPF Centromere protein F CFP Cyan Fluorescent Protein

CFS Common fragile site

CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate CHFR Checkpoint with Forkead and Ring Finger Domain

CHK1 Checkpoint kinase 1

CMG CDC45, MCM2-7 and GINS

CUL2LRR1 Cullin RING ligase 2 associated with LRR1 DAPI 4′,6-diamidino-2-phenylindole

DDK DBF4-dependent kinase (DBF4; DumbBell Forming protein 4)

DDR DNA Damage Response

DMEM Dulbecco´s Minimum Eagle’s Media DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DSB Double-Strand Break

DUB Deubiquitinase

EdU 5-Ethynyl-2′-deoxyuridine

FACS Fluorescence-activated cell sorting FAF1 FAS-associated factor 1

FBS Foetal Bovine Serum

FDA Food and Drug Administration

FSC Forward scatter

g Times gravity

GINS Go-Ichi-Ni-San

H2A Histone H2A

γH2AX Phosphorylated form of histone H2AX at serine 139

H3 Histone 3

H3S10P Phosphorylated form of histone H3 at serine 10 HAUSP Herpes Virus-Associated Ubiquitin Specific Protease HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HHV Human herpes virus

Hr Hour

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HR Homologous recombination HRP Horseradish peroxidase HSV Herpes simplex virus

HTM High Throughput Microscopy ICP0 Infected Cell Polypeptide 0

IgG Immunoglobulin G

iPOND Isolation of Proteins on Nascent DNA JAMM JAB1/MPN/Mov34

KO Knock-Out

LMNA Lamin A

LC-MS Liquid chromatography-Mass spectrometry LiF Leukemia inhibitory factor

M Mitosis

MAPKAPK-2 Mitogen activated protein kinase (MAPK) activated protein kinase 2 MCM-BP Minichromosome Maintenance Complex Binding Protein

MCM3 Minichromosome Maintenance Complex Component 3 MCM7 Minichromosome Maintenance Complex Component 7 MCPIP Monocyte chemotactic protein-induced proteins

MDM2 Mouse Double Minute 2 homolog MEF Mouse Embryonic Fibroblast mESC Mouse embryonic stem cells MiDAS Mitotic DNA repair synthesis

Min Minute

MINDY Motif interacting with ubiquitin-containing DUB family MJD Machado-Joseph disease proteases

mTOR Mechanistic Target Of Rapamycin Kinase

MUS81 MUS81 Structure-Specific Endonuclease Subunit MYT1 Myelin Transcription Factor 1

n Number

NB Nuclear body

Ncz Nocodazole

NEBD Nuclear envelope breakdown

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NHEJ Non-homologous end-joining NP-40 Nonident-P40

NPC Nuclear pore complex

NPLOC4 Nuclear Protein Localization Protein 4 Homolog

OA Okadaic acid

ORC Origin-recognition complex ORI Origin of replication

OTU Ovarian-tumor protease

p21 Cyclin-dependent kinase inhibitor 1 p53 Tumor suppressor protein 53 PBS Phosphate Buffer Saline

PCC Premature chromosome condensation PCNA Proliferating Cell Nuclear Antigen Pen/Strep Penicillin/Streptomycin

PFA Paraformaldehyde

PI Propidium Iodide

PIPES Piperazine-N,N′-bis(2-ethanesulfonic acid) PLK1 Polo like kinase 1

PML Promyelocytic leukemia

POLD1 DNA Polymerase Delta 1, Catalytic Subtunit POLD3 DNA Polymerase Delta 3, Accessory Subtunit PP2A Protein phosphatase 2

PP2A-A Protein phosphatase 2 Scaffold Subunit PP2A-B Protein Phosphatase 2 Regulatory Subunit PP2A-C Protein Phosphatase 2 Catalytic Subunit PRC1 Protein Regulator of Cytokinesis 1 pr-RC pre-Replicative Complex

PTM Post-translational modification RFC2 Replication Factor C, Subunit 2

RNA Ribonucleic acid

RPA Replication Protein A RPE Retinal Pigment Epithelium

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pRb Retinoblastoma protein

RS Replication Stress

RSR Replication Stress Response SAC Spindle Assembly Checkpoint SAE1/Aos1 SUMO activating enzyme 1 SAE2/Uba2 SUMO activating enzyme 2

SAHF Senescence-Associated Heterochromatin Foci

SCFDia2 Skp, Cullin, F-box-containing complex associated with Dia2

SDS Sodium Dodecyl Sulphate

siRNA Small-interference ribonucleic acid ssDNA Single-stranded deoxyribonucleic acid

STAT3 Signal Transducer and Activator of Transcription 3 STUbL SUMO-targeted Ubiquitin Ligase

SUMO Small Ubiquitin-Like Modifier SV40 Simian virus 40

TCEP Tris-2(-carboxyethyl)-phosphine TEAB Triethylammonium bicarbonate

THY Thymidine

TKO Triple Knock-Out

TMRE Tetramethylrhodamine ethyl ester perchlorate Topo I Topoisomerase I

Topo II Topoisomerase II

TRAIP TRAF Interacting Protein UAE Ubiquitin-activating enzyme

Ub Ubiquitin

UBXN3 UBX domain-containing 3

UFD1L Ubiquitin Fusion Degradation Protein 1 Homolog UCH Ubiquitin carboxy-terminal hydrolase

UPS Ubiquitin-proteasome system USP Ubiquitin-specific protease USP7 Ubiquitin Specific Protease 7

v/v volume/volume percentage

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VCP/p97 Valosin Containing Protein

WB Western blot

WEE1 WEE1 G2 checkpoint kinase

WT Wild-Type

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INTRODUCTION

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The cell cycle

The mammalian cell cycle has been traditionally described as a highly regulated sequence of ordered events that lead to chromosome duplication and ultimately to cell division. This classical model, first described in 1953, consists of four independent phases (G1, S, G2 and mitosis (M)) (Figure 1) that are connected in a cyclic manner. The genetic material is replicated during S-phase and chromosome segregation and cell division occur during mitosis. These are the two key events in the cell cycle and are separated by two gaps (G1 and G2) that govern the readiness of cells to enter S- or M-phases respectively.

As a safeguard mechanism, several checkpoints across the cell cycle ensure the proper functioning of all these processes and control the progression during the cell cycle to prevent the transmission of mutations to the next generation of cells. Since the cell cycle regulation is conserved across many eukaryotic species, the use of yeast as a model organism has been essential to study cell cycle.

1.1. Cell cycle regulators: CDKs and cyclins

The progression through the different transitions in the cell cycle is driven by cyclin- dependent kinase (CDK) complexes composed by a CDK catalytic subunit, whose activation requires the binding regulatory subunits, the cyclins (Malumbres and Barbacid, 2009). Even if mammalian cells have around 20 different CDKs (Malumbres et al., 2009), only four of them are known to be involved in the control of the cell cycle: cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) work during interphase, while cyclin-dependent kinase 1 (CDK1, also known as cell division control protein 2 (CDC2)) is the master regulator of mitotic entry (Malumbres and Barbacid, 2009). The rest of CDKs participate in the control of transcription or do not have a clear function assigned yet (Malumbres and Barbacid, 2005). Surprisingly, knock- out models in mice have shown that while CDK2/4/6 play important roles in the cell cycle, CDK1 alone is sufficient to drive the mammalian cell cycle (Santamaria et al., 2007).

Regarding their activating partners, cyclins are synthesized at specific time points of the cell cycle to induce CDK activity at these stages and then degraded by highly specific ubiquitin-mediated proteolysis (Murray, 2004). Briefly, at the beginning of the cell cycle in G1, mitogenic signals inactivate the retinoblastoma protein (pRb) to induce the expression of cyclin D that binds to CDK4 and CDK6 (Malumbres and Barbacid, 2001, Massague, 2004). These CDK4/6-Cyclin D complexes trigger the expression of cyclin E, which subsequently activates CDK2 to initiate S-phase. In late S-phase cyclin A is synthesized and partners first with CDK2 to allow the transition to G2/M and then cyclin A will activate CDK1 at the last stages of this transition. Finally, the key step in G2/M transition is the

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binding of cyclin B1 to CDK1 that induces nuclear envelope breakdown (NEBD) and drives cells through mitosis (Otto and Sicinski, 2017, Hochegger et al., 2008) (Figure 1).

1.2. Moving forward through the cell cycle: The Threshold model

The cell cycle works as a one-way street and its unidirectionality is ensured through the control of three specific transitions: the G1/S transition, mitotic entry and the metaphase-anaphase transition. It has been proposed that feedback loops generate sharp changes in CDK activity (Novak et al., 2007) to enforce the progression in the cycle.

However, this feedback loop mechanism does not explain how CDKs control the temporal order of cell cycle events and when a specific set of proteins is phosphorylated to trigger the next event in the cell. Thus, two alternative views have been proposed for the control of these transitions. First, the cell cycle can be seen as a sequence of reactions where the initiation of every step depends on the completion of the previous one (known as the domino theory). Alternatively, the transitions can be controlled by a biochemical oscillator (the clock theory) (Hartwell and Weinert, 1989), in a model where every CDK/cyclin complex that is activated phosphorylates its targets depending on their specificity.

Although the domino theory has been traditionally favoured, early experiments showing that the fusion of an interphase and a mitotic cell induces a mitotic-like state (Johnson and Rao, 1970) question its validity. On the other hand, experiments in fission yeast showed

Figure 1. The cell cycle. The cell cycle consists of four consecutive and highly regulated phases. The progression through the cell cycle is governed by different CDK/cyclin complexes.

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that it is possible to drive the cell cycle with just one single CDK/cyclin complex, raising doubts about the role that target specificity actually plays in the cell cycle transitions. Later, a threshold model was proposed where an accumulation of CDK activity is seen as the main trigger of cell cycle progression, with the specific modification of target proteins playing a minor role in the process. Accordingly, the progression through cell cycle is driven by the balance between CDKs and their opposite proteins, the phosphatases, and the temporal order of phosphorylations and dephosphorylations depends on the amount of CDK activity required for those modifications (Swaffer et al., 2016, Bouchoux and Uhlmann, 2011, Örd et al., 2019). Although this model can explain why deoxyribonucleic acid (DNA) replication occurs before mitosis, it does not give an answer about the mechanisms of control that prevent a premature increase in CDK activity that may push cells into mitosis too fast.

DNA replication

DNA replication is the process that mediates the copy of the genetic material that will be transmitted to the daughter cells during mitosis, making it a crucial event in the cell cycle. This process can be divided into three phases: during initiation the DNA in the origin of replication (ORI) is unwounded by the pre-replicative complex (pre-RC); then in the

Figure 2. An overview of DNA replication. DNA replication is composed of three phases: DNA initiation, when the helicases are loaded, elongation, when the polymerase copies the genetic material, and termination when replisome

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elongation phase the replication forks duplicate the DNA; and finally at termination converging replication forks meet and the replisome is disassembled (Figure 2).

2.1. DNA replication initiation

In eukaryotic cells, DNA replication initiates from thousands of ORIs distributed along multiple chromosomes in order to duplicate large genomes in relatively short periods of time. To secure that only one single round of genome duplication occurs per cell cycle and to prevent the transmission of additional copies of the genetic material to daughter cells that would lead to genomic instability (Zeman and Cimprich, 2014, Magdalou et al., 2014, Truong and Wu, 2011), DNA replication initiation is established as a two-step mechanism in different phases of the cell cycle (Siddiqui et al., 2013, Skarstad and Katayama, 2013, Blow and Dutta, 2005, Diffley, 2004). The first step, known as licensing, takes place during late mitosis and the onset of G1, when the six-subunit origin-recognition complex (ORC) binds to specific DNA sequences in the ORIs. ORC then recruits adenosine tri-phosphate (ATP)ase cell division cycle 6 (CDC6) and CDC10-dependent transcript 1 (CDT1) (Blow and Dutta, 2005, DePamphilis et al., 2006). These three factors then load the two minichromosome maintenance (MCM) 2-7 helicase complex on each ORI (Evrin et al., 2009, Remus et al., 2009) thereby forming the still inactive pre-RC. Origin licensing is restricted to M and G1 by the phosphorylation of pre-RC components by CDKs (Machida et al., 2005, Liu et al., 2009) and by the proteolysis of CDC6 and CDT1 that prevent the re-replication of the DNA to maintain genome integrity (Piatti et al., 1995, Nishitani et al., 2006). The second step is origin firing and takes place in S-phase. While many origins stay dormant, a subset of previously licensed pre-RCs is activated (fired) (Blow et al., 2011) through the coordinated action of different post-translational modifications (PTMs).

On one hand the phosphorylation of MCM2-7 by Dbf4-dependent kinase (DDK) and other CDKs promotes the loading of cell division cycle protein 45 (CDC45) (Sheu and Stillman, 2006), followed by the recruitment of the four-subunit Go-Ichi-Ni-San (GINS) leading to the formation of the active CDC45, MCM2-7 and GINS (CMG) helicase (Ilves et al., 2010, Moyer et al., 2006, Aparicio et al., 2009). On the other hand, small ubiquitin-like modifier (SUMO)ylation of the MCM complex acts as a novel negative regulator of DNA replication initiation and needs to be removed before cells enter S-phase (Wei and Zhao, 2016). The excess of dormant origins serves as a backup mechanism to be activated under conditions that induce stalled replication forks (Ibarra et al., 2008).

2.2. DNA replication elongation

Once the CMG complex opens the DNA, additional replicative factors are recruited to the ORI to form two active replication forks that progress bidirectionally in the 3’ to 5’

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direction, unwinding and synthesizing DNA during elongation. CMG complexes travel on the leading strand directly associated to DNA polymerase ε while the lagging strand is copied in a discontinuous manner by DNA polymerase δ. Unwinding of the DNA by the forks generates topological stress that is relieved by topoisomerases I or II (Topo I or II) (Zechiedrich and Osheroff, 1990).

2.3. DNA replication termination

In contrast to initiation or elongation, the mechanisms that drive DNA replication termination have remained unexplored, especially in eukaryotic cells, until recent years (Dewar and Walter, 2017). In eukaryotes, similar to bacteria and the simian virus 40 (SV40), the action of Topo II relieves the topological stress generated when two replication forks converge, although this activity is not essential for termination to occur (Espeli et al., 2003, Hiasa and Marians, 1996, Zechiedrich and Osheroff, 1990, Postow et al., 2001, Heintzman et al., 2019). Due to the antiparallel nature of the DNA molecule, when forks converge the CMG helicases encircle different strands and can pass each other to complete the synthesis of the DNA (Fu et al., 2011). Recent studies show that the dissociation of the CMG helicase is a key event in termination, which only occurs after the DNA from two converging forks is fully replicated and ligated (Dewar et al., 2015). At the molecular level, replisome disassembly requires the ubiquitination of replication factors such as minichromosome maintenance complex component 7 (MCM7) (Maric et al., 2014, Moreno et al., 2014), which drives their extraction from chromatin by the recruitment of the valosin containing protein (VCP/p97) segregase, a AAA-ATPase that cooperates with different cofactors to extract ubiquitinated proteins from cellular compartments (Meyer et al., 2012). In the context of DNA replication termination, VCP/p97 recognizes MCM7 ubiquitination through Ubiquitin Fusion Degradation Protein 1 Homolog (UFD1L) and Nuclear Protein Localization Protein 4 Homolog (NPLOC4) adaptors (Maric et al., 2017, Sonneville et al., 2017). In addition, unloading of the MCM complex in late S-phase is facilitated by minichromosome maintenance complex binding protein (MCM-BP) (Nishiyama et al., 2011). The E3 ubiquitin ligase complex that ubiquitinates MCM7 upon DNA replication termination has been identified in S. Cerevisiae (Skp, Cullin, F-box- containing complex associated with Dia2 (SCFDia2)) and in C. elegans and Xenopus (Cullin RING ligase 2 associated with LRR1 (CUL2LRR1)) (Dewar et al., 2017, Sonneville et al., 2017, Maric et al., 2014, Maculins et al., 2015), but not yet in mammals. Finally, after replisome unloading, DNA duplexes are ligated and decatenated (Figure 3).

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Disruption of replisome unloading is toxic for cells. Yeast cells lacking Dia2 accumulate post-termination replisomes where the CMG helicase could not be unloaded in S-phase and stays on DNA until the G1 phase of the next cell cycle (Maric et al., 2014).

This alteration then leads to constitutive activation of the replication checkpoint, sensitivity to DNA-damaging agents and gross chromosomal rearrangements (Blake et al., 2006, Pan et al., 2006). However, in C. elegans deficient for CUL2LRR1 the CMG is unloaded by a second pathway that involves VCP/p97, an additional VCP/p97 cofactor, UBX domain- containing 3 (UBXN3; the worm orthologue of human FAS-associated factor 1 (FAF1)) and the SUMO-protease ULP4, acting as a backup mechanism for the disassembly of the replication machinery in mitosis (Sonneville et al., 2017). Abrogating both pathways by combined knockdown of LRR1 and UBXN3 stabilizes CMG on chromatin until metaphase and is synthetically lethal, again suggesting that replisome unloading is essential for cellular viability (Sonneville et al., 2017). In higher eukaryotes like Xenopus, another E3 ubiquitin ligase, TRAF Interacting Protein (TRAIP), ubiquitinates MCM7 and is involved in removing all replisomes from chromatin before cell division (not only terminated replisomes but also stalled ones) in a process dependent of VCP/p97 (Priego Moreno et al., 2019).

Figure 3. DNA replication termination. From a simple view, when two forks are going to collapse, an ubiquitin E3 ligase targets the MCM7 subunit of the helicase, thus recruiting the segregase VCP/p97 which will remove the ubiquitinated factors from the replisome.

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2.4. Problems during DNA replication: Replication stress

The maintenance of genomic stability is continuously challenged by endogenous or exogenous agents that induce DNA damage, which ultimately can lead to accelerated aging or cancer development (Hanahan and Weinberg, 2000, Burrell et al., 2013). Lesions in the DNA generated difficult the process of DNA replication and can lead to the generation of mutations or genomic alterations. Thus, repairing DNA damage is of vital importance as genetic material must be faithfully duplicated in order to be transmitted to the daughter cells during mitosis. Cells have developed multiple mechanisms to detect and repair different DNA lesions, collectively known as the DNA damage response (DDR) (Kastan and Bartek, 2004, Harper and Elledge, 2007). This response activates specific checkpoints to delay the cell cycle and allow the repair of DNA (Harper and Elledge, 2007).

In order to limit the expansion of the damaged cells or if the damage cannot be repaired the DDR can trigger apoptosis or senescence, mainly through the activation of the tumor suppressor protein 53 (P53) (Harper and Elledge, 2007).

Problems that arise during DNA replication and/or lesions that lead to the stalling of replication forks are known as replication stress (RS). Analogous to the DDR, cells have evolved a RS-response (RSR) that safeguards genome integrity during replication.

Replication fork stalling happens during every S-phase due to the presence of DNA alterations, difficult to replicate regions, conflicts with the transcription machinery or shortage in the supply of nucleotides. In addition, the activation of oncogenes also increases the levels of RS as a consequence of the accelerated rates of cell division. RS is characterized by the presence of high amounts of single-stranded DNA (ssDNA) at the replication fork, caused by the uncoupling of the helicase and the DNA polymerases. A persistent block of the replication fork can give rise to double-strand breaks (DSBs), very dangerous lesions that need to be repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR) (Ciccia and Elledge, 2010).

The RSR follows the hierarchical model of DDR activation where sensor proteins recognize the damage leading to the activation of effector proteins through PTM-mediated signaling pathways. In this case, the triggering signal for RS is ssDNA coated by the replication protein A (RPA) complex, which is then sensed by an ataxia telangiectasia and Rad3 related-ATR interacting protein (ATR-ATRIP) complex, leading to the activation of the kinase activity in ATR. This kinase phosphorylates many substrates, including checkpoint kinase 1 (CHK1), the principal factor of the checkpoint response that is activated by RS (Berti and Vindigni, 2016). The ATR/CHK1 signaling pathway acts at different levels to coordinate DNA replication and cell cycle progression to protect cells

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under the presence of RS. In this sense, the RSR limits global origin firing (by blocking CDC45 loading on chromatin through CHK1 inhibition of CDK2, DDK or Treslin) while promoting local firing of new origins next to the stalled forks (Guo et al., 2015, Boos et al., 2011, Heffernan et al., 2007, Hills and Diffley, 2014) and stabilizing stalled replication forks (Berti and Vindigni, 2016). Finally, the ATR/CHK1 pathway prevents mitotic entry with non- replicated DNA through the inhibitory phosphorylation of cell division cycle 25 (CDC25) by CHK1 and the activation of WEE1 that lead to reduced CDK activity (Mailand et al., 2000, Chen et al., 2003, Peng et al., 1997, Lee et al., 2001).

Mitosis

Once a cell has replicated its genome during S-phase, it will split the genetic material into two daughter cells in during mitosis. This process is divided in five consecutive and morphologically different phases: prophase, prometaphase, metaphase, anaphase and telophase (Musacchio and Salmon, 2007). During prophase chromosomes are gradually condensed, microtubules polymerize from centrosomes and the mitotic spindle starts to form by moving the previously duplicated pair of centrioles to the two poles. Then, NEBD occurs during prometaphase and microtubules connect chromosomes through their kinetochores to the opposite poles of the spindle. In metaphase chromosomes are aligned in the middle of the spindle to form the metaphase plate. Next, in anaphase the microtubules are shortened and separate the chromatids by bringing them to the poles, ensuring that every daughter cell will receive exactly one copy of each chromosome.

During telophase the nuclear membrane is reformed and chromosomes decondensate.

Finally, the cytoplasm is distributed into the two daughter cells during the process of cytokinesis.

3.1. The mitotic trigger is an irreversible cellular switch

As in other transitions of the cell cycle, mitosis is controlled by a complex phosphorylation pathway mediated by mitotic kinases such as Aurora A (AURKA), Aurora B (AURKB) or polo like kinase 1 (PLK1) and phosphatases like CDC25 and protein phosphatase 2 (PP2A). These kinases and phosphatases control essential mitotic events such as centrosome duplication and maturation, spindle formation, chromosome condensation and cytokinesis (Barr et al., 2004, Barr and Gergely, 2007, Archambault and Glover, 2009, Carmena and Earnshaw, 2003, Ruchaud et al., 2007, Takaki et al., 2008, Bollen et al., 2009, Barr et al., 2011).

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Above this complex network of kinases and phosphatases, CDK1 is the master regulator of mitotic entry (Nurse, 1990, Enserink and Kolodner, 2010) and blocking its activity leads to the arrest of cells in G2 before mitosis (Vassilev et al., 2006). Thus, the activation of CDK1 needs to be tightly regulated. The first step in the activation of CDK1 is its binding to cyclin B1 (Santamaria et al., 2007). This cyclin is synthesized at the end of S-phase and G2, and unlike other cyclin proteins, is located in the cytoplasm during interphase to prevent the activation of CDK1 before mitosis (Pines and Hunter, 1991). A second level of regulation involves the modification of CDK1 by different PTMs such as phosphorylation, SUMOylation or acetylation (Xiao et al., 2016, Deota et al., 2019). Among these PTMs, the equilibrium between the activating phosphorylation in threonine 161 (T161) by cyclin-dependent kinase 7 (CDK7) (Fisher and Morgan, 1994, Mäkelä et al., 1994) and the inhibitory phosphorylations in threonine 14 (T14) and tyrosine 15 (Y15) by the kinases WEE1 and myelin transcription factor 1 (MYT1) (Heald et al., 1993, Parker and Piwnica-Worms, 1992, Gould and Nurse, 1989, Mueller et al., 1995) controls the switch of the CDK1/cyclin B1 complex from an inactive to an active state. These inactivating phosphorylations can be removed by the family of phosphatases CDC25 (CDC25A, B and C) (Perry and Kornbluth, 2007, de Gooijer et al., 2017). A third level of regulation is the establishment of a feedback loop where CDK1 can activate itself through positively regulating CDC25 and inhibiting WEE1 and MYT1, inducing a rapid amplification of CDK1 activity (Mueller et al., 1995, O'Farrell, 2001). Thus, the activation of CDK1 relies on the formation of the CDK1-Cyclin B complex, the establishment of an active pattern of PTMs and then the activation of a feedback loop to amplify the activation of CDK1. Similar to CDK1, the protein phosphatase PP2A acts as a master phosphatase that controls the phosphorylation of CDK1 and other mitotic targets. The inhibition of PP2A with Okadaic acid (OA) activates CDK1 activity and induces entry into mitosis (Tournebize et al., 1997, Saurin, 2018, Mochida et al., 2009) (Figure 4). Importantly, the true trigger that determines when mitosis starts is still an open question.

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3.2. From mitotic entry to anaphase

The onset of mitosis is set by the activation and nuclear translocation of the CDK1/CyclinB1 complex induced by a reduced activity of WEE1, MYT1 and PP2A and high activity of CDC25. CDK1 targets hundreds of proteins inducing a cascade of phosphorylations that promote key mitotic events. For example, CDK1-dependent phosphorylation of the nuclear lamina initiates NEBD (Peter et al., 1990, Ward and Kirschner, 1990). Another important target of CDK1 is the anaphase promoting complex/cyclosome (APC/C), an ubiquitin ligase complex that controls the progression through mitosis and subsequently chromosome segregation (Kelly et al., 2014, Chang et al., 2014, Van Voorhis and Morgan, 2014, Brown et al., 2014). Active CDK1 phosphorylates the APC/C complex and cadherin 1 (CDH1), leading to its substitution with cell division cycle 20 (CDC20) and to the subsequent ubiquitination and degradation of securin. The loss of securin releases separase, a protease that is essential for the separation of sister chromatids (Uhlmann et al., 2000, Uhlmann, 2001, Waizenegger et al., 2000, Hauf et al., 2001) and to progress to anaphase.

3.3. The APC/C complex and mitotic exit

Besides facilitating the separation of sister chromatids, the APC/C complex also controls the exit from mitosis by eliciting a sharp decrease in CDK1 activity. When both kinetochores from each chromosome are properly attached to the spindle and the spindle assembly checkpoint (SAC) is satisfied, anaphase proceeds and APC/C degrades cyclin

Figure 4. Regulation of CDK1. CDK1 is regulated by the action of the inhibitory kinases WEE1 and MYT1, the activating phosphatase CDC25 and its counteracting phosphatase PP2A. Once active, it is translocated to the nucleus to trigger mitosis.

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B1 leading to the inactivation of CDK1 (King et al., 1995). In case of an incorrect kinetochore attachment, the SAC acts as a safeguard mechanism inactivating the APC/C complex to prevent securin and cyclin B1 degradation, thus maintaining a pre-anaphase mitotic state until the problem is solved (Musacchio and Salmon, 2007). The degradation of cyclin B1 is essential for mitotic exit and a cyclin B1 mutant that cannot be ubiquitinated leads to sustained CDK1 activity and arrest in mitosis. At the end of mitosis, APC/C interacts again with CDH1 to induce the degradation of mitotic proteins and the phosphatase PP2A is re-activated. The role of PP2A is supported by mutations in its regulatory subunit PP2A-B55, that induce abnormal anaphase resolution (Mayer-Jaekel et al., 1993). Together, APC/C and PP2A coordinate the exit from mitosis and allow cells to complete their division.

Are DNA replication and mitosis really independent?

Traditionally, S-phase and mitosis have been considered two independent phases in the cell cycle separated by G2, the time when the cell prepares for cell division. However, 20 years ago a work from Charles D. Laird lab reported, for the first time, evidences for very late DNA replication during the early stages of mitosis, indicating that DNA replication and mitosis might actually overlap (Widrow et al., 1998). Further work supporting a view where the end of S-phase and the entry into mitosis might be functionally coupled has been published in recent years.

4.1. DNA replication during mitosis (MiDAS)

Organisms with large genomes carry DNA loci that are difficult-to-replicate such as common fragile sites (CFSs). While replication of these regions usually takes place in late S-phase, recent reports have shown that RS can delay the replication of CFS until early mitosis in a process known as mitotic DNA repair synthesis (MiDAS). MiDAS is mediated by the recruitment of the endonuclease complex MUS81-EME1-SLX4 to CFSs, which promotes DNA synthesis through the formation of a DNA polymerase complex with translesion synthesis polymerases and a non-catalytic subunit of the polymerase δ complex (POLD3) (Bhowmick et al., 2016, Minocherhomji et al., 2015). MiDAS also requires the action of the E3 ubiquitin ligase TRAIP in human cells (Sonneville et al., 2019), consistent with experiments in C. elegans showing that replisome disassembly during mitosis is also driven by TRAIP (Deng et al., 2019, Priego Moreno et al., 2019). These evidences raise the possibility that DNA replication and mitosis are not really independent processes.

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4.2. The S/M checkpoint and premature mitotic activation

The previous results show that DNA replication can take place during mitosis.

Conversely, a slew of experiments demonstrate that mitotic signaling activity can also be unleashed during S-phase. Fusion experiments of interphase and mitotic cells (Johnson and Rao, 1970), inhibition of phosphatases (Ajiro et al., 1996) or WEE1 (Aarts et al., 2012, Sorensen and Syljuasen, 2012, Ruiz et al., 2016), deletion of wee1 (Russell and Nurse, 1987) or a constitutively active mutation of CDK1 (Szmyd et al., 2019) all induce premature mitotic activation in S-phase. In all cases, this activation leads to premature chromosome condensation (PCC) and ultimately cell death by mitotic catastrophe. Mechanistically, premature CDK1 activity drives chromosome pulverization which is thought to be executed by several endonucleases (Duda et al., 2016, Aarts et al., 2012). Consequently, it is important to prevent mitotic entry before DNA replication has been fully completed, supporting the existence of a S/M checkpoint that restricts CDK1 activity as a mechanism to safeguard genomic stability (Hartwell and Weinert, 1989, Enoch and Nurse, 1991).

However, how this checkpoint works is still not fully understood.

4.3. Coordinating DNA replication and mitosis

Despite the recent studies shedding light into how DNA replication termination occurs, and all the knowledge acquired about how mitotic kinases are activated, many questions about the connection between these two events remain unanswered. What is the real nature and length of G2? How do cells sense they have to activate their mitotic program?

In other words, what is the actual signal that triggers mitosis? These questions have been outstanding for over 20 years and while the threshold model can explain the order of the events of the cell cycle it does not justify the regulation of their timing (McHedlishvili et al., 2015).

If DNA replication and mitotic activation were independent processes, the end of DNA replication would not have an influence on when mitosis is triggered. However, analysis of the substrates of mitotic kinases has revealed that their phosphorylation occurs already in G2 (Ly et al., 2017). Actually, the activation of mitotic kinases is set during the S/G2 transition when the phosphorylation of their targets can be already detected, suggesting a possible link between DNA replication and mitotic activation (Lemmens et al., 2018, Akopyan et al., 2014). Taking into account that the activation of mitotic kinases can be forced in S-phase, these data raise the question of what triggers mitotic kinases at the S/G2 transition. Conversely, it suggests that their activation is blocked during a non- perturbed cell cycle until cells reach the end of S-phase.

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In this sense, a recent paper from the Lindqvist lab showed that preventing DNA replication initiation prematurely activates CDK1 and PLK1 (Lemmens et al., 2018), suggesting that DNA replication itself might be limiting mitotic activity. Additionally, a similar phenotype can be found after the inhibition of the two main kinases in the RSR, ATR or CHK1 (Lemmens et al., 2018, Saldivar et al., 2018). One of the main functions of the RSR is the inhibition of CDK to prevent the progression into mitosis with unreplicated DNA (Cimprich and Cortez, 2008, Lopez-Contreras and Fernandez-Capetillo, 2010).

During a non-perturbed S-phase, the basal activity of ATR and CHK1 could be enough to suppress the activation of mitotic kinases. Based on these findings, it has been recently proposed that DNA replication acts as a brake to prevent mitotic activation (Lemmens and Lindqvist, 2019). Interestingly, work in Xenopus has shown that increasing CDK1/cyclin B1 activity can also induce the ubiquitination of the CMG helicase by TRAIP and the disassembly of the replisome (Deng et al., 2019). Thus, DNA replication can limit the activation of mitotic kinases and conversely, the activation of CDK1 promotes the disassembly of the replisome from chromatin.

Ubiquitination and SUMOylation: and overview

The regulation of most cellular processes and in particular the control of the cell cycle heavily relies on signaling pathways mediated by PTMs, modifications deposited in the side chain of specific aminoacids in the target proteins. These PTMs can be small functional groups as in the case of phosphorylation or even small proteins as ubiquitin or SUMO, which can modulate protein levels, activity, localization or signaling. In recent years the modification of the replication machinery by ubiquitin and SUMO has been shown to play very important roles in the control of DNA replication and in the RSR.

5.1. The ubiquitin-proteasome signaling pathway

Ubiquitin conjugation is an essential mark in eukaryotic cells (Komander and Rape, 2012, Pickart and Eddins, 2004) since it targets proteins for degradation by the proteasome in what is known as the ubiquitin-proteasome system (UPS). Thus, protein ubiquitination is essential for cellular proteostasis and constitutes the central pathway in the quality control of proteins in the cell by inducing the degradation of misfolded or damaged proteins (Hochstrasser, 2009). Further, it also plays a role in the dynamic control of protein levels mediating the degradation of proteins that need to be destroyed as is the case for cyclins during the cell cycle (Hershko and Ciechanover, 1998).

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Ubiquitin deposition is mediated by a cascade of three reactions that ultimately leads to the covalent attachment of the small protein ubiquitin to a lysine residue in the target protein through the formation of an iso-peptide bind between the amino group in the lysine and the C-terminus of the ubiquitin molecule. First an E1 ubiquitin-activating enzyme (UAE) activates ubiquitin in the presence of ATP. Once activated, ubiquitin is transferred to a cysteine residue of the E2 ubiquitin-conjugating enzyme, which acts as a carrier to deliver it to the E3 ubiquitin-ligase enzyme. This ubiquitin ligase confers substrate specificity to finally add the ubiquitin molecule on the target protein (Hochstrasser, 2009).

The ubiquitin system allows a wide variety of modifications from the addition of one single ubiquitin molecule (mono-ubiquitination) to the modification of multiple lysines with one ubiquitin molecule (multi-mono-ubiquitination) or the formation of ubiquitin chains through the conjugation of additional ubiquitin molecules on specific lysine residues of the ubiquitin protein (poly-ubiquitination) (Haglund and Dikic, 2005, Hochstrasser, 2009, Ye and Rape, 2009). The type and topology of these modifications determines the outcome of the ubiquitination event in the target protein.

Ubiquitination is reversed by the action of deubiquitinases (DUBs), ubiquitin specific proteases that release ubiquitin molecules from their targets (Nijman et al., 2005, Clague et al., 2013). There are different subclasses of DUBs based on their protease domains (Ubiquitin-specific proteases (USPs), ubiquitin carboxy-terminal hydrolases (UCHs), ovarian-tumor proteases (OTUs), Machado-Joseph disease proteases (MJDs), the monocyte chemotactic protein-induced proteins (MCPIPs), and the novel motif interacting with ubiquitin-containing DUB family (MINDY), all belonging to zinc metalloproteases and JAB1/MPN/Mov34 (JAMMs) belonging to zinc metalloproteases (Mennerich et al., 2019).

Among DUBs, USPs are the largest group, possibly due to the evolutionary diversification of their functions in concert with the evolution of E3 ubiquitin ligases (Semple et al., 2003).

As indicated before, ubiquitination plays multiple roles in the cell cycle including the control of DNA replication termination, the degradation of cell cycle regulators and the activation of some of the checkpoints (King et al., 1995, Hauf et al., 2001, Maric et al., 2014, Moreno et al., 2014). Noteworthy, the control of cellular proteostasis has become an important therapeutic approach for cancer treatment, leading to the development of inhibitors against different components of the UPS such as the proteasome, E3 ubiquitin ligases or DUBs (Nicholson et al., 2007, Orlowski and Kuhn, 2008, Shi and Grossman, 2010, Hoeller and Dikic, 2009). Some of these compounds, such as the proteasome inhibitors Bortezomib or Carfizomib, have already been approved by the U.S. Food and Drug Administration (FDA).

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5.2. The SUMOylation pathway

Other small proteins similar to ubiquitin also act as PTM, including the small ubiquitin- like modifier (SUMO) (Mahajan et al., 1997). Protein SUMOylation is mediated by a similar enzymatic cascade to ubiquitination but using specific enzymes: a dimeric E1 SUMO- activating enzyme (formed by the two subunits SAE1/Aos1 and SAE2/Uba2), a unique E2 SUMO-conjugating enzyme (Ubc9) and a number of E3 SUMO-ligases that will catalyze the binding of SUMO to the lysine residue of the target protein, conferring selectivity of the process (Gareau and Lima, 2010). However, and in contrast to ubiquitination, the E2 SUMO-conjugating enzyme Ubc9 can directly transfer SUMO to the final substrate in the absence of an E3 SUMO-ligase. Additionally, eukaryotes express three SUMO proteins (SUMO1, SUMO2 and SUMO3), although SUMO2 and SUMO3 share 97% of their sequence identity and have indistinguishable functions, thus being usually referred as SUMO2/3 (Saitoh and Hinchey, 2000, Tatham et al., 2001).

Ubiquitin and SUMO are not independent pathways and in fact, on many proteins there is a competition between the ubiquitination and SUMOylation on the same target residues. Furthermore, SUMO can act a as a signal for a subsequent ubiquitination (Hendriks et al., 2014), facilitating a cross-talk between both pathways (Sha et al., 2019, Schimmel et al., 2008).For example, SUMO2/3 chains recruit SUMO-targeted ubiquitin (Ub) ligases (STUbLs) to promote their poly-ubiquitination and degradation.

SUMOylation is reported to target thousands of proteins (Hendriks and Vertegaal, 2016), which are frequently on chromatin or on certain regions of the nucleus such as promyelocytic leukemia (PML) nuclear bodies (PML-NBs) (Lallemand-Breitenbach and de Thé, 2018). SUMO controls multiple cellular processes including nuclear transport, transcription, chromatin remodeling, DNA repair, cell cycle, and ribosomal biogenesis (Xiao et al., 2016, Wei and Zhao, 2016, Bergink and Jentsch, 2009, Jackson and Durocher, 2013, Gareau and Lima, 2010, Stielow et al., 2008, Ulrich, 2012). Interestingly, SUMO expression is frequently up-regulated in human cancers where it has been proposed to be important for the growth of transformed cells (Seeler and Dejean, 2017). Due to its relevance in so many important pathways, SUMOylation is essential in most organisms from yeast to mammals.

During DNA replication SUMOylation regulates many steps, being necessary for the loading of the MCM complex and inhibiting CDK1, among other functions (Xiao et al., 2016, Wei and Zhao, 2016). Moreover, some years ago we revealed the existence of a SUMO/ubiquitin switch during DNA replication (Lopez-Contreras et al., 2013), whereby replicating DNA is enriched in SUMOylated factors and depleted in ubiquitinated proteins

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and the opposite occurs in mature chromatin. In addition, we revealed that the USP7 deubiquitinase is important to maintain this micro-environment (Lecona et al., 2016).

The deubiquitinase USP7

The Ubiquitin Specific Protease 7, USP7, also known as Herpes Virus-Associated Ubiquitin Specific Protease (HAUSP) is a deubiquitinase that was identified as a protein associated with the E3-ubiquitin ligase Human Herpes Virus (HHV) Infected Cell Polypeptide 0 (ICP0), in herpes simplex virus (HSV) (Everett et al., 1997). USP7 has been reported to be a ubiquitous protein which targets multiple proteins and regulates many processes on chromatin, from DNA replication to transcription and repair (Faustrup et al., 2009, Alonso-de Vega et al., 2014, Colleran et al., 2013, Lecona et al., 2015, Lecona et al., 2016, Song et al., 2008, Zhu et al., 2015, van der Horst et al., 2006).

6.1. USP7 and the regulation of the MDM2/P53 pathway

Soon after its identification it was discovered that USP7 plays a key role in the regulation of the tumor suppressor P53, whose proteins levels are mainly controlled by its ubiquitination mediated by the E3 ligase mouse double minute 2 homolog (MDM2) (Li et al., 2002, Li et al., 2004, Cummins and Vogelstein, 2004). USP7 binds, deubiquitinates and stabilizes both MDM2 and P53, and the net result of its activity depends on the cellular context. In the absence of stress, USP7 prevents the self-ubiquitination of MDM2, leading to its stabilization, the poly-ubiquitination of P53 and its degradation. In response to stress, the action of USP7 is shifted to preferentially deubiquitinate P53 thereby promoting its accumulation while MDM2 ubiquitinates itself and is degraded (Figure 5). Based on these observations USP7 inhibitors have been developed as anticancer agents, with the idea of stabilizing P53 to induce cell death. The search for USP7 inhibitors has further expanded in the last years (Zhang and Sidhu, 2018), yielding new and more selective small molecules active against USP7 (Gavory et al., 2018, Kategaya et al., 2017, Lamberto et al., 2017). Importantly, USP7 knock-out is lethal in mice and this lethality is not rescued by deletion of P53 (Kon et al., 2010, Kon et al., 2011), in contrast to the rescue of the MDM2 knock-out mice after P53 deletion (Montes de Oca Luna et al., 1995). Moreover, the DNA damage induced by USP7 inhibitors is independent of P53 (Lecona et al., 2016).

Collectively, all these data show that while P53 might be a very important substrate of USP7 the toxicity of USP7 inhibitors cannot be solely ascribed to the regulation of the MDM2/P53 axis.

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6.2. USP7 and DNA replication

USP7 has been shown to play different roles during DNA replication. First, USP7 was described to regulate the unloading of the MCM complex from chromatin at the end of S- phase by acting on MCM-BP (Jagannathan et al., 2014). Moreover, USP7 also regulates the levels of Geminin, a negative regulator of the origin licensing factor Cdt1 (Hernandez- Perez et al., 2017). The stability of other replication factors or proteins involved in the RSR is controlled by USP7, including CHK1, claspin or Rad18 among others (Faustrup et al., 2009, Alonso-de Vega et al., 2014, Martin et al., 2015, Zlatanou et al., 2016). Further, USP7 has also been suggested to deubiquitinate proliferating cell nuclear antigen (PCNA) to control the translesion synthesis pathway (Qian et al., 2015, Kashiwaba et al., 2015).

In this regard, we previously described how USP7 is essential to regulate a SUMO/ubiquitin switch during DNA replication (Lopez-Contreras et al., 2013). Our work revealed that USP7 is a SUMO dependent deubiquitinase that limits the ubiquitination of replication factors during DNA replication. Interestingly, USP7 also removes ubiquitination from SUMO chains and USP7 inhibition leads to the ubiquitination and displacement of SUMOylated proteins from the replisome (Lecona et al., 2016) (Figure 6). Based on these data, we proposed that USP7 inhibition could be used as a model to mimic DNA replication termination in mammalian cells (Lecona and Fernandez-Capetillo, 2016).

Figure 5. Model of P53 regulation by MDM2 and USP7.

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6.3. USP7 and mitosis

USP7 has also been reported to regulate the ubiquitination of different mitotic proteins.

For instance, USP7 depletion was shown to delay early events in mitosis, thereby stabilizing cyclin B1 and also destabilizing the mitotic E3 ubiquitin ligase Checkpoint with Forkhead and Ring Finger Domain (CHFR). As a result AURKA, a substrate for CHFR, is stabilized and accumulates leading to the appearance of multipolar spindles (Giovinazzi et al., 2013). Another work related USP7 to the mitotic exit checkpoint through its interaction with the budding uninhibited by benzimidazoles 3 homolog (BUB3), one of the key SAC components. USP7 depletion destabilizes BUB3 leading to genomic instability, abnormal chromosome segregation and micronuclei accumulation. Interestingly, this phenotype occurs in a P53 independent manner (Giovinazzi et al., 2014). In addition to that, tumor suppressor P53 Binding Protein 1 (53BP1) phosphorylation by PLK1 or AURKA leads to its stabilization by USP7, promoting its binding to the centromere protein F (CENPF), a regulator of proper kinetochore attachment (Yim et al., 2017).

In light of the recent data suggesting that active DNA replication may act as a brake for mitotic entry, and taking into account the functions of USP7 during S phase and mitosis it would be interesting to understand what is the role for this deubiquitinase in the transition from S-phase to mitosis.

Figure 6. Effect of USP7 inhibition on the replisome.

USP7 prevents SUMOylated factors from being ubiquitinated and displaced from the replisomes.

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OBJECTIVES

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During the course of this Doctoral Thesis we set the following objectives:

1. To further evaluate if USP7 inhibition triggers DNA replication termination.

2. To investigate the existence of a potential link between DNA replication and mitotic entry.

3. To determine if a premature activation of the mitotic signaling cascade contributes to the toxicity of USP7 inhibitors.

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MATERIALS AND METHODS

Referencias

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