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Thesis

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On start and pause of replication fork

SHYIAN, Maksym

Abstract

In my PhD thesis I describe the work I've done with my colleagues in Professor David Shore's Lab that led to the next 3 main conclusions. (1) Conserved protein Rif1 is a negative regulator of DNA replication initiation in budding yeast. Rif1 counteracts DDK-dependent activatory phosphorylation at Mcm4 and Sld3 replication initiation proteins by recruiting protein phosphatase PP1 (ScGlc7) through its RVxF/SILK motifs. (2) Replication-restraining activity of Rif1 is important for the maintenance of genome integrity. Cells lacking Rif1 have elevated origin activation in early S phase at ribosomal RNA gene tandem repeat array (rDNA), which leads to destabilization of the array and dependency on DSB repair/fork maintenance factors MRX and Mms22-Ctf4 for survival. (3) Replisome pausing factors Tof1-Csm3 promote pausing at RFB independently of replisome accessory helicase Rrm3. Replication fork pausing at proteinaceous barriers (RFBs) depends on DNA lagging strand synthesis machinery and topoisomerases I and II.

SHYIAN, Maksym. On start and pause of replication fork. Thèse de doctorat : Univ.

Genève, 2018, no. Sc. 5255

DOI : 10.13097/archive-ouverte/unige:110396 URN : urn:nbn:ch:unige-1103966

Available at:

http://archive-ouverte.unige.ch/unige:110396

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Département de biologie moléculaire Professeur David Shore

On Start and Pause of Replication Fork

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Maksym SHYIAN

d’

Odessa (Ukraine)

Thèse n° 5255

Genève Atelier ReproMail

2018

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1 TABLE OF CONTENTS ...p1 ABSTRACT...p3 RÉSUMÉ...p5 ACKNOWLEDGEMENTS ...p7 INTRODUCTION ...p9 DNA replication and genome integrity ...p9 A need for a helicase and a swivel to duplicate chromosomes ...p9 DNA replication initiation ...p9 Origins of replication and their licensing ...p11 Replicative helicase and its activation ...p11 Two-level foolproof control of origin firing by DDK and CDK ...p12 Limiting initiation factors for origin firing ...p13

Spatiotemporal replication coordination in normal conditions and under damage ...p13 Rif1 ensures the replication timing program ...p15 Open questions ...p16 Replication fork progression at fork barriers (RFB) ...p17 Replisome Progression Complex ...p18 Accessory helicases ...p18 Replication fork pause and its modulation by Tof1-Csm3 and Rrm3/Pif1...p19 Replisome progression and DNA topology ...p22 Open questions ...p22 Replication initiation and fork pausing at ribosomal RNA gene (rDNA) repeats array ...p23

rDNA array composition in various organisms with focus on yeast ...p23 rDNA replication ...p24 RFBs at rDNA ...p25 rDNA integrity ...p26 rDNA and yeast aging ...p27 Open questions ...p27 Goals of the thesis ...p28 RESULTS ...p29 PART I: Replication initiation is restricted by Rif1-Glc7 and N-terminal region of Mcm4 ...p29

Cover page for (Mattarocci et al., 2014) ...p29 Rif1 controls DNA replication timing in yeast through the PP1 phosphatase Glc7 (Mattarocci et al., 2014) ...p30-p53 N-termini of Mcm5 and Mcm4 are the main inhibitors of DNA replication initiation at DDK-controlled step ...p54 PART II: Replication initiation and pausing at rDNA is the primary mediator of Rif1 effects on genome integrity in budding yeast ...p58

Yeast Rif1 plays a role in genome integrity ...p58 Cover page for (Shyian et al., 2016) ...p62 Budding yeast Rif1 controls genome integrity by inhibiting rDNA replication (Shyian et al., 2016) ...p63-p99 Genetic interaction of RIF1 with SAE2 is mediated through Fob1-RFB ...p100

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2 Rif1 promotes rDNA silencing and recruitment of Cdc14 and Brn1

...p100 Rif1 is phosphorylated in G2/M and upon DNA DSB induction via CDK-DDK and Mec1- Rad53 ...p101 PART III: Replication fork pausing involves DNA lagging strand synthesis machinery and topoisomerases 1/2 ...p107 Mechanisms of replication fork pausing at proteinaceous barriers in budding yeast

...p107-p130 Introduction ...p108 Results ...p109 Tof1-Csm3 promote fork pausing at RFBs independently of Rrm3 helicase ...p109 Mapping the replication fork pausing/blocking by replisome component ChIP....p111 Forward genetic screen to discover genes responsible for RFB tolerance ...p112 Disruption of DNA lagging strand synthesis diminishes the fork pause at Fob1-RFB ...p114 1D-2D gels candidate screen for factors mediating replication fork pausing ...p118 Simultaneous disruption of topoisomerase 1 and 2 alleviates fork pausing at Fob1- RFB ...p119 Discussion ...p120 DNA lagging strand metabolism promotes replisome pausing ...p122 Top1 and Top2 cooperatively promote replication fork pause at Fob1-RFB ...p122 Materials and methods ...p124 References ...p126 DISCUSSION ...p131 DNA replication initiation control by conserved protein Rif1 ...p131

Rif1-PP1 targets relevant for DNA replication ...p131 Molecular mechanism of selectivity in Rif1 action at origins ...p132 Rif1 is a phosphoprotein regulated by CDK-DDK, Mec1-Rad53 and Glc7

...p134 Genome stability control by conserved protein Rif1 ...p135 Replication fork pause at proteinaceous barriers regulation by conserved complex Tof1- Csm3 ...p137 REFERENCES ...p140

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3 ABSTRACT

DNA replication occurs in 3 main stages: initiation, elongation and termination.

Replication is initiated at specific locations along yeast chromosomes, called origins. It proceeds through a sequence of reactions of inactive helicase loading around origin DNA (licensing step) and helicase activation and replication fork establishment (firing step). The helicase activation occurs via activatory phosphorylation by essential kinases DDK and CDK.

The DDK-dependent phosphorylation alleviates the intrinsic inhibitory activity of Mcm4 N- terminus and leads to recruitment of firing factors Sld3/7 and Cdc45 to the origins. Although many potential origins are licensed, not all of them are used as replication initiation sites in a given S phase. Moreover, origins differ in their efficiency and the relative time along the progression of S phase when they usually fire, called replication timing. Until recently, the molecular mechanism maintaining this strict pattern of origin firing in space and time (spatiotemporal program) was not known. We and others showed that evolutionary conserved protein Rif1 is the master regulator of the replication timing. Mechanistically, Rif1 counteracts the DDK-dependent phosphorylation of helicase component Mcm4 and firing factor Sld3 by recruiting PP1 protein phosphatase Glc7, thereby precluding replication initiation. Moreover, Rif1 itself is a target of CDK/DDK and Glc7, which appear to modulate Rif1 function. Forward genetic screen for replication inhibitors of the DDK-regulated origin firing step yielded exclusively Rif1 and N-terminal parts of Mcm4 and Mcm5 proteins, proving them as the main regulators of this initiation stage.

The loss of spatiotemporal replication program upon disruption of Rif1 turned out to compromise genome integrity through rDNA tandem repeat array. Namely, we observed advancement of origin firing at rDNA locus accompanied by fragilization of this locus that led to increased sickness induced by endogenous and exogenous DNA damage in strains lacking replication fork maintenance/repair factors. The negative effects of Rif1 loss could be suppressed by removal of site specific polar replication fork barrier (RFB) created by Fob1 protein at rDNA repeats. Alleviation of the replication pause from the replisome side by removal of the fork protection complex Tof1-Csm3 also suppressed sickness in cells lacking both RIF1 and MRE11.

The elongation step of the replication is interrupted by fork slowing down/pausing at structural and protein barriers. Protein RFBs are sensed by Tof1-Csm3, evolutionary conserved component of the replisome (TIMELESS-TIPIN in mammals). We employed

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4 forward genetic screen searching for factors contributing to rDNA stability in the absence of Tof1-Csm3 and 1D-2D gel based candidate screen to broaden our understanding of the fork pausing phenomenon. We discovered that replisome pausing involves DNA lagging strand synthesis machinery and topoisomerases 1/2.

Results supporting the above models, additional data and discussions are presented in my thesis.

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5 RÉSUMÉ

La réplication de l'ADN se produit en 3 étapes: l’initiation, l’élongation et la terminaison. La réplication est initiée à des endroits spécifiques le long des chromosomes de la levure, appelés origines de réplication. Ce phénomène débute par la séquence de réactions de chargement de l'hélicase inactive autour de l'ADN d'origine (étape de ‘licensing’

- l'autorisation) puis l'activation de l'hélicase et l'établissement de la fourche de réplication (étape de ‘firing’ – ou le déclenchement) prennent place. L'activation de l'hélicase se fait par phosphorylation activatrice par les kinases essentielles DDK et CDK. La phosphorylation dépendante de DDK allège l'activité inhibitrice intrinsèque de l'extrémité N-terminale de Mcm4 et conduit au recrutement des facteurs de déclenchement Sld3/7 et Cdc45 aux origines. Bien que de nombreuses origines potentielles soient autorisées, elles ne sont pas toutes utilisées comme sites d'initiation de réplication dans une phase S donnée. De plus, les origines diffèrent quant à leur efficacité et le temps relatif le long de la progression de la phase S lorsqu'elles se déclenchent habituellement, appelé le programme spatiotemporel de la réplication. Jusqu'à récemment, le mécanisme moléculaire conservant ce modèle strict d'origine dans l'espace et le temps (programme spatiotemporel) n'était pas connu. En parallèle avec d’autres chercheurs, nous avons montré que la protéine conservée évolutive Rif1 est le régulateur principal du programme temporel de la réplication. Mécaniquement, Rif1 contrecarre la phosphorylation dépendante de DDK du composant hélicase Mcm4 et du facteur de déclenchement Sld3 en recrutant la protéine phosphatase PP1 (Glc7), empêchant ainsi l'initiation de la réplication. De plus, Rif1 elle-même est une cible de CDK/DDK et Glc7, qui semblent moduler la fonction Rif1. Le criblage génétique direct des inhibiteurs de réplication de l'étape d’activation régulée par DDK a exclusivement fourni des parties de Rif1 et N-terminales des protéines Mcm4 et Mcm5, apportant ainsi des preuves additionnelles qu’il s’agit bien des principaux régulateurs de cette étape d'initiation.

La perte du programme de réplication spatiotemporelle lors de la perturbation de Rif1 s'est avérée compromettre l'intégrité du génome par l'intermédiaire du réseau de répétition en tandem de l'ADNr. Notamment, nous avons observé l’avancement de l'activation des origines au locus ADNr accompagné de la fragilisation de ce locus conduisant ainsi à une maladie accrue induite par des dommages endogènes et exogènes de l'ADN dans des souches manquant de facteurs de maintenance/réparation de fourche de réplication. Les effets négatifs de la perte de Rif1 pourraient être supprimés par l'élimination de la barrière

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6 de réplication polaire spécifique (RFB, ‘Replication Fork Barrier’) à un site créée par la protéine Fob1 au niveau des répétitions d'ADNr. L'allégement de la pause de réplication du côté du réplisome par la suppression du complexe de protection des fourches Tof1-Csm3 a également supprimé la maladie dans les cellules qui ne possèdent ni RIF1 ni MRE11.

L'étape d'allongement de la réplication est interrompue par un ralentissement ou une pause de la fourche aux barrières structurales et protéiques. Les RFB protéiques sont reconnues par Tof1-Csm3, composant conservé évolutif du réplisome (TIMELESS-TIPIN chez les mammifères). Nous avons utilisé un criblage génétique pour identifier les facteurs contribuant à la stabilité de l'ADNr en l'absence de Tof1-Csm3 ; et un criblage à base de gel 1D-2D pour élargir notre compréhension du phénomène de la pause de la fourche de réplication. Nous avons découvert que la pause de réplisome implique un mécanisme de synthèse des brins retardé d'ADN et des topoisomérases 1/2.

Les résultats soutenant les modèles ci-dessus, des données supplémentaires et des discussions sont présentés dans ma thèse.

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7 ACKNOWLEDGEMENTS

I thank David Shore for inviting me to join his laboratory and for giving me the opportunity to work with him. I am grateful to all the present and past members for friendly and inspiring working environment, for unparalleled movie and rumors discussions during lunch times together. I thank the Department of Molecular Biology for exciting Chapitres Choisis and other seminars. I thank the kitchen workers for the glassware and media. I thank my colleagues from 3rd and adjacent floors for tolerating my singing-while-working all these years. I thank Stefano Mattarocci for proposing me to join him in investigating Rif1 action, for many shared experiments and discussions. I would like to thank Dogus Altintas for urging me to ‘’sit down and think it over’’ and to Stefano Mattarocci for equally eagerly warning me

‘’not to overthink’’. I thank Andreja Moset Zupan for being a great student. I thank Benjamin Albert for reagents, protocols and discussions. I thank Jessica Bruzzone for help with DNA sequencing libraries preparation, Marcus Smolka and Michael Lanz for discussions, suggestions and phsophoproteome experiments. Alessandro Bianchi, Anne Donaldson, Jim Haber, Lorraine Symington for reagents (strains and plasmids). I thank Daniel Dilg and Benjamin Albert for correcting the French version of this thesis’s Abstract. I thank Benoît Kornmann for sharing details of causative mutation identification by a single back-cross WGS strategy. I thank Philippe Pasero and Thanos Halazonetis for evaluating my PhD thesis and defense.

I thank my wife Nataliia Serbyn for her constant support and understanding, both when things worked and when they did not; for help with protocols, reagents, strains and discussions and everything. I thank my family for boundless worrying and caring about me. I thank my friends for sharing live moments.

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8 Epigraph

“It is good to have an end to journey toward; but it is the journey that matters, in the end.”

― Ursula K. Le Guin, The Left Hand of Darkness

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9 INTRODUCTION

DNA replication and genome integrity

Faithful duplication of genetic information is essential for existence of all the known living things. In the three main domains of life, Archaea, Bacteria and Eukarya, this genetic information is stored as DNA. According to the Central Dogma of Molecular Biology the information stored in the DNA is executed through the sequential processes of transcription and translation so that the living cells can metabolize, grow and reproduce. During the cell division the DNA of the mother cell should be duplicated in its entirety and transferred to the daughter cells. Similar to transcription and translation, DNA replication occurs in three main steps: initiation, elongation and termination. The initiation step is believed to be the most crucial and tightly regulated, as once a cell enters S phase and starts replication there is no way back.

A need for a helicase and a swivel to duplicate chromosomes

The specific DNA structure where the duplication of parental double helix occurs is called replication fork due to its appearance. The assembly of all the proteins at the replication fork is called replisome. The main components of replisome are helicases that unwind DNA and polymerases that synthesize the new strands. The DNA polymerases act on single-stranded DNA (ssDNA) substrates. Therefore, in order to duplicate a DNA molecule the double helix has to be unwound by the action of helicases. As the two strands are topologically interlinked and pass each other every ca. 10.5 bp they should be also unlinked with the help of topoisomerases (Duguet, 1997). At the end of replication, converging forks meet and the replisomes are dis-assembled with help of dedicated ubiquitin-dependent machinery (Dewar and Walter, 2017) and the sister chromatids are disentangled by topoisomerase 2 (Baxter and Diffley, 2008).

DNA replication initiation

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10 DNA replication starts at specific locations, or origins (Scheme 1). Most bacteria have one well defined origin of replication, while eukaryotes have many (Leonard and Mechali, 2013). Budding yeast has well-defined sequence-specific origins of replication, which together with the Awesome Power of Yeast Genetics turned this organism in the best model to study the fundamental principles of organization and regulation of chromosomal duplication in eukaryotes (Duina et al., 2014).

Scheme 1: Origins and their licensing and firing. Many origins assemble inactive Mcm2-7 helicase on them in M/G1 phases (origin licensing). Some origins are destined to fire early in S phase, while other will fire later or don’t fire at all. Rif1 is the main factor ensuring this spatiotemporal program of origins firing. Cells lacking Rif1 therefore exhibit firing of both early and late origins in early/middle S phase (where cells can be arrested by addition of HU – hydroxyurea).

Yeast origins, also known as ARS (Autonomously Replicating Sequence) were initially discovered in yeast by their ability to initiate replication of DNA when inserted into an episomal vector (Bell and Labib, 2016). Later, a plethora of genome-wide methods, such as CsCl (Cesium Chloride) density-gradient centrifugation followed by microarrays, ORC and MCM chromatin immunoprecipitation (ChIP), BrdU immunoprecipitation, ssDNA mapping etc, was used to create a near-full list of all the active, dormant and potential origins of the yeast genome, available for instance through OriDB (Siow et al., 2012). In total, budding

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11 yeast genome contains ca. 300-1000 origins, depending on the method of detection (Leonard and Mechali, 2013).

The vertebrate genome is on average 500x times larger than the budding yeast genome and it is expected to contain therefore up to 100.000 origins (Creager et al., 2015).

However, vertebrate origins seem to lack a well-defined consensus sequence, apart from a tendency to have G nucleotides at certain positions (OGRE – origin G-reach elements) (Leonard and Mechali, 2013). The identification of the origin consensus sequences might be also complicated in vertebrates due to a proposed relocation of the Mcm2-7 (replicative helicase) complexes along the chromosomes by transcription machinery (Gros et al., 2015;

Powell et al., 2015).

In eukaryotes the replication initiation from origins occurs in 2 separate and sequential steps. First, the origins are ‘licensed’ – the reaction of loading of inactive Mcm2-7 helicase onto all the origins that occurs in late M and G1 cell cycle phases (Bell and Labib, 2016).

Second, some of the loaded helicases are activated in the so called ‘firing’ step by additive activity of essential cell cycle kinases DDK and CDK (Bell and Labib, 2016).

Origins of replication and their licensing

Yeast origin sequence contains an ACS motif (for ARS Consensus Sequence), which is a recruitment sequence for ORC complex (Origin recognition complex) composed of 6 proteins Orc1-6 (Leonard and Mechali, 2013). In budding yeast ORC complex is localized to the ARS throughout the cell cycle. Only in M/G1 phase the ORC complex recruits Cdc6 protein and loads Mcm2-7/Cdt1 complex onto the origin DNA. Two Mcm2-7 complexes assembled with their N-terminal parts head-to-head encircling double stranded DNA of an origin constitute the so called ‘Mcm4-6 double hexamer’ which is an inactive form of replicative helicase (Deegan and Diffley, 2016).

Replicative helicase and its activation

The active replicative helicase, or holoreplicative complex, is called CMG reflecting its composition of Cdc45 protein, one Mcm2-7 hexamer and GINS complex (11 polypeptides in total). Leading strand polymerase epsilon tightly associates with CMG and stimulates the helicase activity; therefore the holoreplicative complex is also called sometimes CMGE (Zhou et al., 2017). The eukaryotic replicative helicase encircles ssDNA and has 3’-5’ translocation

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12 polarity (opposite polarity of bacterial replicative helicase DnaB). Replicative helicase activation is therefore a reaction of transformation of the inactive Mcm2-7 double hexamer encircling dsDNA into an active CMG(E) encircling ssDNA.

It’s currently emerging that this activation process in not a one-step reaction, but might be further subdivided into a sequence of reactions (Douglas et al., 2018). An elegant study showed that upon helicase activation the origin DNA is melt and one ssDNA is extruded through the Mcm2-5 gate of the Mcm ring (Samel et al., 2014).

It is worth noting that some of the basic assumptions in the DNA replication field could be revised, as it was recently demonstrated that Mcm proteins in the active CMG helicase are traveling N-terminus first at the replication fork, - contrary to previous belief (Douglas et al., 2018; Georgescu et al., 2017).

With this new understanding of the Mcm polarity at replication fork it becomes evident that upon inactive Mcm2-7 double hexamer activation, two CMG complexes should first pass each other and move in opposite directions after (Georgescu et al., 2017). This passage reaction is proposed to involve activity of both CMG and therefore may constitute a quality control to ensure correct establishment of bidirectional replication (Georgescu et al., 2017).

Two-level foolproof control of origin firing by DDK and CDK

Replicative helicase assembly/activation and replication fork establishment are mediated through phosphorylation of multiple origin firing components by two essential cell cycle kinases DDK (Dbf4-dependent kinase) and CDK (Cyclin-dependent kinase). DDK phosphorylates the serine/threonine residues in NSD domains (for N-terminal serine/threonine-rich domain) of Mcm2/4/6, in which it is assisted by CDK and probably Mec1 (Randell et al., 2010). There are two main models in the field explaining the DDK- mediated step of Mcm2-7 activation. In the first model, proposed by Stillman and colleagues, the N-terminal NSD domain of the Mcm4 protein in the double hexamer negatively regulates (opposes) helicase activation and this inhibitory activity is annihilated by DDK-dependent phosphorylation (Sheu and Stillman, 2010). The second model, proposed by Diffley and colleagues, doesn’t imply an inhibitory effect, rather it suggest that the phosphorylated N-termini of Mcm4/6 serve as landing pad for helicase activating firing factors Sld3/7, which are readers of this modification (Deegan et al., 2016). In any case, the

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13 net effect of DDK-dependent phosphorylation is the recruitment of Sld3/7 and Cdc45 proteins to the Mcm2-7 (Tanaka and Araki, 2013). Through some still unresolved steps, this recruitment is accompanied by double hexamer dissociation into two hexamers, the origin DNA melting and single CMG helicase transfer onto ssDNA (Tanaka and Araki, 2013). In parallel, additional firing factors are associated with the CMG, largely due to the bridging activity of CDK-dependent phosphorylation. DDK’s essential step is an induction of architectural switch in the Mcm2-7 double hexamer, as a point mutation in proline 83 of Mcm5 protein, mcm5-bob1 mutation, bypasses the necessity of DDK catalytic activity (Jackson et al., 1993).

The essential role of CDK in DNA replication is to create a bridge between Sld2 and Sld3 through Dpb11, presumably in order to recruit polymerase epsilon to the helicase (Tanaka et al., 2007; Zegerman and Diffley, 2007). Indeed, Dpb11 serves as a linker protein in this reaction, using its BRCT domains to grip simultaneously Sld2-p (interacting with pol epsilon) and Sld3-p (on the CMG helicase) (Tanaka et al., 2007; Zegerman and Diffley, 2007).

Limiting initiation factors for origin firing

Importantly, many of the firing factors are present in the cell in limiting amounts at G1/S transition (Mantiero et al., 2011; Tanaka et al., 2011). In other words, the number of these proteins is much lower than the number of potential origins of replication.

Consequently, the sheer amount of these factors would suffice therefore to initiate replication only on a subset of origins at the beginning of S phase (Mantiero et al., 2011;

Tanaka et al., 2011). Consistently, overexpression of these factors - so called SSDD or SSDDCS settings (abbreviation of the first letters of the factors Sld2/3, Dbf4, Dpb11 and Cdc45, Sld7) – leads to advancement of origin firing time of normally late origins (Mantiero et al., 2011;

Tanaka et al., 2011). Most of the firing factors (with the exception of Cdc45) are dissociating after the replication fork establishment and could be re-used to activate other origins later in S phase.

Spatiotemporal replication coordination in normal conditions and under damage

Origins differ in their efficiency (a percent probability of an ARS to initiate replication in a given S phase) and timing (relative time during S phase when an ARS tend to initiate replication) (Leonard and Mechali, 2013; Rhind and Gilbert, 2013). Indeed, origins of

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14 replication do not fire all at the same time. Some tend to fire earlier into S phase (or simply

‘early’ origins – ca. 250 origins in budding yeast) while other fire later (‘late’ origins – ca. 250 in budding yeast) or normally don’t fire at all (dormant origins). Centromere-proximal origins are typically the earliest, while telomeric origins are the latest (Bell and Labib, 2016; Rhind and Gilbert, 2013). Many of the late and dormant origins therefore rarely get a chance to fire and are passively replicated by forks coming from early origins (Bell and Labib, 2016; Rhind and Gilbert, 2013). `Therefore, late origins tend to exhibit a generally lower efficiency than their early counterparts (Bell and Labib, 2016; Rhind and Gilbert, 2013). The molecular basis for the timing difference and its biological purpose is being debated. As noted above, the currently dominant model in the field is the ‘limiting firing factors’ model, where the number of firing factors could not sustain firing of all the potential origins at the same time in the cell. Therefore these firing factors act at the earliest origins first and then get recycled during the S phase – sequentially activating later and later origins. Under this model, slowing down the replication fork rate would give enough time to recycle these factors and fire most of the origins, - something indeed observed in vivo (Alvino et al., 2007). The preferential association of the limiting factors to early origins vs late first in S phase might be mediated by relative chromatin accessibility of the origins to the limiting factors. Indeed, it was shown that chromatin modifying enzymes histone acetylases and deacetylases can modulate the firing time of origins both in cis (Vogelauer et al., 2002) and in trans (Yoshida et al., 2014).

Significantly however, most of the genome-wide effects of the Sir2 and Rpd3 deacetylases on the origin firing were shown to be indirect in trans due to competition of genomic origins with numerous ribosomal RNA genes (rDNA) origins of replication for the limiting firing factors (Yoshida et al., 2014). Another mechanism to specify early origins is preferential DDK recruitment through Dbf4 subunit via forkhead transcription factors Fkh1/2 (Fang et al., 2017; Knott et al., 2012) or specific recruitment to centromeres via kinetochore component Ctf19 (Natsume et al., 2013).

As for the biological role of replication timing program, it was proposed to: preclude dNTP exhaustion, leave spare late/dormant origins for rescue of stalled/collapsed replication forks, regulate gene dosage along S phase, tune mutation levels along the genome or just be a byproduct of genome architecture and limiting firing factors (Rhind and Gilbert, 2013).

Upon DNA damage or exhaustion of dNTP pools in S phase, the cell induces DNA replication checkpoint – a pathway slowing down the S phase and mobilizing the cell

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15 metabolism for efficient dealing with replication stress. The RPA-coated single-stranded DNA at active replication forks is the main signal of the replication stress. It activates the essential Mec1 (ATR in vertebrates) kinase, which in turn phosphorylates the effector kinase Rad53 with the help of replication checkpoint adaptor Mrc1 and Tof1-Csm3. The main physiological outcomes of the replication checkpoint activation are: overexpression and over activation of the essential ribonucleotide reductase complex (responsible for dNTP supply), inhibition of late origins firing, phosphorylation of replisome and DNA repair components in order to limit replication fork degradation (Hustedt et al., 2013). It is believed that by doing so the cell tries to slow down the S phase and finish the replication with the help of existing replication forks by reinforcing them without activating additional origins thereby precluding further aggravation of the replication factors shortage.

The replication checkpoint exploits the origin activation bottleneck by modulating activity of limiting firing factors. Specifically, activated Rad53 phosphorylates and inactivates Dbf4 and Sld3, therefore precluding further origin firing (Lopez-Mosqueda et al., 2010;

Zegerman and Diffley, 2010).

Rif1 ensures the replication timing program

The spatiotemporal program of origin activation is mediated by a conserved protein Rif1 (Mattarocci et al., 2016). In the absence of Rif1 protein the late origin firing is greatly advanced in budding (Dave et al., 2014; Hiraga et al., 2014; Mattarocci et al., 2014) and fission yeast (Hayano et al., 2012), fruit fly (Sreesankar et al., 2015), frog (Alver et al., 2017), mouse (Sukackaite et al., 2017), and human cells (Yamazaki et al., 2012). The Rif1 protein appears to act through a different pathway than the replication checkpoint, as Rif1-defficient cells are proficient in slowing down the S phase under replication stress (Alver et al., 2017;

Peace et al., 2014) and exhibit even stronger Rad53 phosphorylation than Rif1-containing cells (Shyian et al., 2016). The molecular mechanism by which Rif1 acts is just starting to emerge. Several laboratories discovered that Rif1 is a regulatory/targeting/adaptor factor for an essential protein phosphatase PP1 (Glc7 in budding yeast) (Dave et al., 2014; Hiraga et al., 2014; Mattarocci et al., 2014). The current model in the field has it that Rif1 recruits PP1 to the origins to reverse the DDK-dependent activatory phosphorylation of the Mcm2-7 complex, primarily at the N-terminus of Mcm4 protein. Recent structural (Mattarocci et al., 2017; Moriyama et al., 2018), biochemical (Mattarocci et al., 2017; Moriyama et al., 2018)

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16 and chromatin distribution (Hafner et al., 2018; Kanoh et al., 2015) studies support a model that Rif1 might physically interact with chromosomes thanks to the DNA-binding property of its HEAT repeats (Mattarocci et al., 2017; Moriyama et al., 2018). Moreover, Rif1 affects the 3D architecture of chromosomes (Foti et al., 2016). It is reasonable to assume therefore that Rif1 acts either in cis by recruiting PP1 to regulated origins and/or regulates accessibility of origins to limiting firing factors in trans by changing chromosomal architecture and affecting competition between origins. In budding yeast Rif1 is recruited to telomeres via interaction with telomere-binding protein Rap1 (Hardy et al., 1992); consistently, the telomere-proximal origins show the greatest sensitivity to Rif1 in the cell (Hafner et al., 2018).

Furthermore, Rif1 appears also to contain a DDK/CDK-regulated switch for positive feed-back loop of origin activation in S phase (Dave et al., 2014; Hiraga et al., 2014). Rif1 physically interacts with DDK and the Rif1-PP1 interaction in budding and fission yeast is negatively controlled by DDK/CDK-dependent phosphorylation of serine/threonine residues around the RVxF/SILK motifs involved in PP1 (Glc7) binding. In fly embryos Cdk1 was shown to inhibit association of Rif1 itself with chromatin (Seller and O'Farrell, 2018). Thus, the build-up of DDK and CDK activities at the G1/S transition and in S phase would lead to phosphorylation of Rif1 N-terminus, PP1 dissociation and avalanche-like origin firing (Hiraga et al., 2014).

Apart from Rif1, DNA polymerase theta is the only other non-firing factor implicated in replication timing control (Fernandez-Vidal et al., 2014). Loss of polymerase theta in human cells leads to increased MCM proteins association with chromatin in G1 cell cycle phase, advancement in firing of some late origins and delay of some early origins. Interestingly, similarly to Rif1 (Chapman et al., 2013; Escribano-Diaz et al., 2013; Zimmermann et al., 2013), polymerase theta is also promoting DNA DSB repair via NHEJ pathway (Mateos- Gomez et al., 2015). However, the mechanism of polymerase theta action in DNA replication timing and whether it is Rif1-related is currently unknown.

Open questions:

- What is the exact molecular mechanism of the origin firing regulation by N-terminal regions of Mcm2/4/6 proteins?

- How Rif1 protein recognizes origins in budding yeast?

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17 - Are there other mechanisms, in addition to Rif1, to directly regulate replication timing?

- Do DDK/CDK function on the active CMG also after its incorporation into replisome during the elongation and termination steps?

- What is the mechanism of action of polymerase theta in replication timing?

Replication fork progression at fork barriers (RFB)

After its activation by the firing reaction, the CMG helicase sheds some of these firing factors, recruits additional components, like polymerases, replicative clamp PCNA with its loader RFC, accessory helicases and so on and starts the journey along the chromosome. On average the replication forks in yeast are bound to travel a distance of approximately 20 kbp before merging with a converging fork (Pasero et al., 2002). It’s important to ensure that all the forks are fused before the cell enters into mitosis and attempts to condense and segregate the chromosomes (Scheme 2).

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18 Scheme 2: Elongating replisome and protein barriers. Replication forks pause at proteinaceous barriers (labeled ‘Stop’ signs here) due to presence of Tof1-Csm3 at replisome. Rrm3 helicase (‘sweepase’) or converging forks (on the right of the figure) help to rescue the paused fork. MRX complex (green dumbbells) helps to maintain the paused fork presumably by precluding its collapse (Bentsen et al., 2013; Tittel-Elmer et al., 2009) and promoting reassembly of replisome (Hashimoto et al., 2011).

Replisome Progression Complex

The seminal study by Labib and colleagues (Gambus et al., 2006) used proteomic MS analysis of a CMG component and identified a set of factors tightly associated with active helicase – the Replisome Progression Complex (RPC). In addition to CMG, RPC contains Mcm10, Ctf4, Mrc1, Tof1-Csm3, FACT and Top1. The function of the RPC components is just starting to emerge. For instance, Mcm10 is necessary for helicase activation initial origin DNA unwinding and pol alpha recruitment (Douglas et al., 2018); Ctf4 is a trimeric hub mediating CMG helicase interaction with polymerase alpha and also multiple accessory factors, like: Dpb2 subunit of pol epsilon, Dna2 endonuclease/helicase, Chl1 helicase, Tof2 rDNA-associated protein and perhaps other factors (Samora et al., 2016; Villa et al., 2016).

FACT (FAcilitates Chromatin Transactions) is a histone chaperon complex mediating chromatin turnover by replication and transcription (Formosa, 2008). Mrc1 helps to recruit pol epsilon to the CMG and cooperates with Tof1-Csm3 in mediating replication checkpoint and accelerating replication (Yeeles et al., 2017).

RPC also associates less strongly with DNA polymerases alpha and epsilon and SCF- Dia2 complex (De Piccoli et al., 2012). Furthermore, proteomic analysis of the nascent chromatin by iPOND or NCC methods (Cortez, 2017) led to identification of dozens and even hundreds of additional factors associated with replication forks. Future studies will reveal what are the roles of these factors and how/when they are recruited to the replication fork.

Accessory helicases

In addition to the main replicative helicase, CMG, replisome also contains so called

‘accessory’ helicases of the PIF1 family – Rrm3 and Pif1 in budding and Pfh1 in fission yeast.

These two helicases have opposite polarity to that of CMG, namely they translocate along

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19 ssDNA in 5’-3’ direction and therefore are believed to be able to operate on the lagging strand template thereby assisting CMG in fork progression through difficult-to-replicate regions (Ivessa et al., 2003). Indeed, Rrm3 helicase assists replication progression through thousands of protein-mediated replication fork barriers (Ivessa et al., 2003), while Pif1 helicase is largely acting on the uncanonical DNA structures, like G-quadruplex (G4) forming sequences (Paeschke et al., 2011). Pif1 is also involved in the processing of Okazaki fragments by a long-flap pathway together with Dna2 (Balakrishnan and Bambara, 2013).

Both Rrm3 and Pif1 were shown to be recruited to the replisome by interaction with replicative clamp PCNA (Buzovetsky et al., 2017).

Replication fork pause and its modulation by Tof1-Csm3 and Rrm3/Pif1

Replication fork progression is not uniform throughout the genome and the replisome tends to slow down at some regions. These pauses occur either at uncanonical DNA structures (trinucleotide repeats, inverted repeats, G4 forming sequences etc.) or at sites of tight binding of some proteins (Gadaleta and Noguchi, 2017; Mirkin and Mirkin, 2007;

Tourriere and Pasero, 2007). The protein replication fork barriers (or RFBs) occur at rDNA (Fob1 protein binding sites), telomeres (Rap1 binding sites), silent mating type loci HML and HMR (ORC complex at dormant origins of these loci), tRNA genes (RNA polymerase III transcription pre-initiation complex - PIC), centromeres (centromere binding factors - CBF).

The protein RFBs temporarily or permanently block advancement of the replisome when it approaches either from both sides (non-polar barriers, as at centromeres) or only from one side (polar barriers, as at Fob1-RFB). Thousands of RFBs could be visualized in the yeast strains lacking accessory helicase Rrm3 (Ivessa et al., 2003) by 2-dimensional agarose gel electrophoresis (2D gels) (Brewer and Fangman, 1988) or chromatin immunoprecipitation (ChIP) of replisome components (Azvolinsky et al., 2009; Sekedat et al., 2010). The pausing can be either short-lasting, like at centromeres, or much longer, like at Fob1-RFB. In the latter case, the paused replication fork is usually blocked for as long as it takes for the converging fork to reach the RFB from another side (Brewer and Fangman, 1988). Because of this, some RFBs tend to overlap with replication termination zones (RTZ), as detected with Bromodeoxyuridine (BrdU) incorporation and 2D gel methods (Fachinetti et al., 2010; Ivessa et al., 2003) and to some extent with Okazaki fragment mapping method (Osmundson et al., 2017).

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20 Belonging to the same PIF1 family, the accessory helicases Rrm3 and Pif1 cooperate in assisting replication fork progression through protein RFBs of Y’ telomere elements (Anand et al., 2012) and tRNA genes (Osmundson et al., 2017). At Fob1-RFB, perplexingly, loss of Rrm3 and Pif1 has opposite effects: rrm3Δ cells exhibit stronger pausing while pif1Δ cells, surprisingly, have weaker block (Ivessa et al., 2000). It’s possible that weakening of Fob1-RFB in pif1Δ cells is mediated by stronger recruitment/activity of Rrm3, as double mutant rrm3Δ pif1Δ is similar to rrm3Δ (Ivessa et al., 2000).

Replication fork pause at protein barriers is positively regulated by a conserved pair of proteins Tof1-Csm3 (in budding yeast) (Calzada et al., 2005; Hodgson et al., 2007; Mohanty et al., 2006; Tourriere et al., 2005), Swi1-Swi3 (in fission yeast) (Dalgaard and Klar, 2000;

Krings and Bastia, 2004) and TIMELESS-TIPIN (in vertebrates) (Akamatsu and Kobayashi, 2015). As of today, Tof1-Csm3 and their homologs where not shown to possess any catalytic activity and are thought to work on replisome via protein-protein and protein-DNA interactions (Leman and Noguchi, 2012; McFarlane et al., 2010). Absence of Tof1-Csm3 leads to loss of pausing at Fob1-RFB, tRNA genes, and centromeres (Calzada et al., 2005; Hodgson et al., 2007; Mohanty et al., 2006; Tourriere et al., 2005), Swi1-Swi3 are necessary for pause at rDNA RFBs and mating type locus in fission yeast ) (Dalgaard and Klar, 2000; Krings and Bastia, 2004). TIMELESS-TIPIN promote pause at rDNA RFB of human cells (Akamatsu and Kobayashi, 2015). Despite the evolutionary conservation of Tof1-Csm3 complex, the mechanism of its action is not known. The prevalent model in the field suggests though that Tof1-Csm3 promote the pausing by inhibiting Rrm3 helicase at replication fork (Mohanty et al., 2006). However, in vitro studies showed that TIMELESS protein alone and in complex with TIPIN are able on their own to modulate the activity of main replication factors (Cho et al., 2013). Namely, TIMELESS inhibits MCM complex activity but at the same time stimulates activities of DNA polymerases alpha, delta and epsilon (Cho et al., 2013). These in vitro effects appear to be consistent with in vivo observations that Tof1, together with Mrc1 (another component of RPC), mediate coupling of the replisome to the site of DNA synthesis under replication stress conditions (Katou et al., 2003). Tof1-Csm3 form a complex with Mrc1 (Bando et al., 2009; Nedelcheva et al., 2005) and together ensure the high rate of replication in vitro (Yeeles et al., 2017) and replication checkpoint activation in vivo (Crabbe et al., 2010). Mrc1 accelerates more the replication fork than Tof-Csm3 (Hodgson et al., 2007; Yeeles et al., 2017) and has stronger contribution to replication checkpoint activation

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21 (Crabbe et al., 2010). However, Mrc1 is not necessary for the replication pause at protein RFBs (Hodgson et al., 2007; Tourriere et al., 2005). Tof1-Csm3 and Mrc1 assist the replication fork progression through uncanonical DNA structures (Voineagu et al., 2008; Voineagu et al., 2009), rather than mediating the pause at these DNA-only sequences.

Therefore, it is reasonable to anticipate that the positive role of Tof1-Csm3 in replisome pausing could be independent of the general fork acceleration and replication checkpoint stimulation effects mediated jointly by Mrc1 and Tof1-Csm3. Whatever the mechanism of replisome pausing at protein RFBs by Tof1-Csm3 is, it should involve recognition of disparate protein barriers and modulation of the replication progression rate.

DDK kinase is a putative interactor of Tof1-Csm3 (Murakami and Keeney, 2014) in budding yeast meiotic cells and of Swi1-Swi3 in fission yeast mitotic cells (Shimmoto et al., 2009). Recently, it was shown that DDK promotes fork pausing at Fob1-RFB by promoting Tof1 recruitment to chromatin and CMG phosphorylation (Bastia et al., 2016).

Interestingly, not all the protein barriers are created equal (Table 1). Engineering of the bacterial polar replication fork barrier Tus/Ter system into the yeast cell unexpectedly showed that it is independent of both Tof1-Csm3 and Rrm3 (Larsen et al., 2014). The reason behind this independence is not known, but might be related to the ‘mousetrap’ mechanism operating in this case, where DNA unwinding (even by non-biological mechanical force) leads to flipping of a conserved cytosine from the Ter sequence into a pocket on Tus protein and establishes a strong barrier to further DNA unwinding (Berghuis et al., 2018).

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22 Table 1. Summary of main studied replication fork barriers and their regulation. S.c – Saccharomyces cerevisiae; S.p – Schizosaccharomyces pombe; H.s – Homo sapiens. n/a – data not available.

Replisome progression and DNA topology

It is expected from the topological point of view, that advancing replication fork lead to formation of positive supercoils in front of the replication fork. Positive supercoils before the replisome can be resolved by topoisomerases 1 and 2 in eukaryotes (Pommier et al., 2016).

Alternatively, the torsional stress could be released by fork rotation and transferred into a topological interweaving of the sister chromatids behind the fork (Pommier et al., 2016). It was proposed recently that Tof1-Csm3 components of the RPC are acting to inhibit the fork rotation (Schalbetter et al., 2015).

Open questions:

- Is there a difference in replisome composition/modifications at the forks arising from early vs late and dormant origins?

- How the multiple accessory factors are recruited to the replisome?

- Is the replication fork speed actively adjusted depending on loci and physiological conditions?

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23 - What is the ‘default’ situation for a fork at RFBs: pause or bypass? In other words, which of these two pathways, pause or bypass, is primary and which is secondary?

- Is there a mechanism detecting damage in the template DNA specifically in front of the fork and directing the repair machinery and/or slowing the replication fork before it runs into the damage?

Replication initiation and fork pausing at ribosomal RNA gene (rDNA) repeats array

rDNA array composition in various organisms with focus on yeast

The genes coding for the RNA part of the ribosomes (rRNA) in eukaryotes are present in multiple copies organized in tandem repeat arrays (Scheme 3). In humans there are around 350 repeats organized in clusters on 5 acrocentric chromosomes (Kobayashi, 2014) while budding yeast have just one array of ca. 150 repeats on the right arm of chromosome XII called RDN1 locus. Each repeat of 9.1kb in length contains a 35S rRNA gene (which is transcribed and processed into 18S, 5.8S and 25S rRNA) and a small 5S rRNA gene, divergently transcribed by RNA polymerase I and III respectively. These two genes are separated by intergenic sequences IGS1 and IGS2, also called NTS1 and NTS2 (for non- transcribed sequences; however this terminology is obsolete, as both IGS1 and 2 give rise to a set of regulatory non-coding transcripts (Kobayashi and Ganley, 2005; Vasiljeva et al., 2008).

The IGS1 region contains a programmed replication fork barrier created by sequence- specific DNA-binding protein Fob1 (Fob1-RFB), 35s rRNA transcription termination site and bidirectional non-coding promoter E-pro (Kobayashi, 2014). The IGS2 sequence contains the promoters of 35S and 5S rRNA gene and the rDNA origin of replication rARS.

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24 Scheme 3: rDNA locus in budding yeast. Ribosomal RNA coding genes are tandemly repeated in an array of around 150 copies on the right arm of chromosome XII (rDNA locus) and packed into nucleolus inside the cell nucleus. As each repeat contains potential origin of replication (rARS), the rDNA locus harbors around 1/3 of all the potential yeast origins. rARS have low firing efficiency (ca. 20%), which is ensured by joint action of Sir2 and Rif1. Each repeat also contains Fob1-RFB pausing rightward moving (telomere proximal direction) replication forks.

rDNA replication

Each rDNA repeat possesses a potential origin of replication rARS, which have a low efficiency of firing ca. 20%, so that only 1/5 of these origins fire in an array in each cell cycle (Brewer and Fangman, 1988). The origins in the rDNA array are not firing randomly but in clusters of 2-3 origins (Pasero et al., 2002). Moreover, activated rARS are usually located downstream from active rRNA genes (Muller et al., 2000). The activity of rARS is intimately correlating with the length of the rDNA array, as both increasing or decreasing of its activity either by DNA sequence variation (Ganley et al., 2009; Kwan et al., 2013) or by change in replication initiation protein factors activity (Ide et al., 2007; Salim et al., 2017; Shyian et al., 2016) leads to instability of the array and change in its size. Because of the strength of the

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25 polar replication fork barrier in the rDNA repeats, the array replication is essentially unidirectional, similarly to that at telomeres.

RFBs at rDNA

Each rDNA repeat also contains a polar Fob1-RFB just downstream from the 35S rRNA gene. This barrier is created by a sequence-specific binding of Fob1 to closely spaced RFB1-3 sequences (Takeuchi et al., 2003) of which two are specifically potent, called Ter1-2 (Brewer and Fangman, 1988). The molecular details of this interaction are not known, but it is proposed that two molecules of Fob1 could bind simultaneously to the RFB sequence (Takeuchi et al., 2003). Fob1 molecules from one repeat can interact with those of another repeats thereby establishing ‘chromosome kissing’ (Choudhury et al., 2015).

The polar replication fork barrier at rDNA downstream from the rRNA genes is a conserved feature present in most organisms with rDNA arrays studied to date (Bastia and Zaman, 2014). The nature of the protein creating the barrier and the number of pause sites is however different. Fission yeast, for instance, have 4 pause/termination sites: Ter1 is created by Sap1, Ter2-3 by Reb1 while RFP4 appears to be a result of replication encounters with transcribing RNA polymerase I. Human rDNA barrier is created by the binding of TTF-1 (Akamatsu and Kobayashi, 2015), which serves as transcription termination factor for RNA polymerase I, – structural and functional orthologue of the fission yeast Reb1. Tof1-Csm3 and their homologs are essential for the pause at all of these barriers with exception of SpRFP4. Importantly, in the absence of Rrm3 helicase the replication forks in budding yeast rDNA repeats start to exhibit additional pausing at 5S rRNA gene, rARS, rRNA transcription termination site and both sides of Fob1-RFB (Ivessa et al., 2000).

What is the biological function of the polar Fob1-RFB at rDNA? There are two main models to explain the conservation of the barrier mechanism. According to the older model, Fob1-RFB precludes the replication forks from entering into an adjacent rRNA repeat in a head-on orientation with its transcription, thereby preventing deleterious collision of DNA replication machinery with RNA polymerase I complex (Bastia and Zaman, 2014). However, deletion of FOB1 gene is not only viable but even confers resistance to certain genotoxic agents (Shyian et al., 2016). Moreover, the replication-transcription collisions are not observed in the fob1Δ cells with rDNA array of physiological size (Takeuchi et al., 2003). The collisions become detectable only if the rDNA array is shortened down to 20 copies so that

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26 each rRNA gene becomes highly transcribed (Takeuchi et al., 2003). The cells with short rDNA array lacking Fob1 protein are exquisitely sensitive to DNA damaging agents and this effect is RNA polymerase I-dependent (Ide et al., 2010). However it was not assessed whether re-expression of Fob1 in the cells with short rDNA array will alleviate the sensitivity to genotoxic agents (by preventing replication-transcription collisions).

The second model posits that Fob1-RFB is a regulatory mechanism to maintain number of rDNA repeats (Kobayashi, 2014) and perhaps adjust its size in accordance with metabolic conditions. This model builds on the fact that Fob1 is responsible for the high level of mitotic homologous recombination (HR) at the rDNA locus. It is proposed that the replication forks at the Fob1-RFB fuel HR by directly recruiting HR machinery or by collapsing into DSBs, which in turn are repaired through HR. Indeed, it would be expected from a repetitive array to be shortened gradually with time due to HR-dependent events like single strand annealing (SSA); so a mechanism to restore repeat number might be evolutionary selected for.

Moreover, Fob1-RFB was also shown to mediate not only formation of the circular episomal excised copies of rDNA – extrachromosomal rDNA circles (ERC) but to also re-capture them back into the array (Mohanty et al., 2009). As some ERC form even in the cells lacking Fob1 (Burkhalter and Sogo, 2004), the array could also lose repeat through this pathway, if not for the Fob1-dependnet re-capturing. This model speculates that the Fob1-dependent recombination could also serve to modulate the size of the array depending on the metabolic growth requirements. This was not shown yet, but it’s worth noting that yeast isolates from different natural sources have different rDNA array size (Kwan et al., 2013). It is also reasonable to assume that the presence of stalled forks or DSBs may serve to fuel homogenization of the rDNA repeats’ sequences by gene conversion.

rDNA integrity

rDNA array is a vulnerable locus, because of several features. First, it is repetitive, which renders it a good substrate for homology-directed repair processes. Second, it’s highly replicated with potential for replication-transcription collision or R-loop induced damage.

Third, the Fob1-RFB is instrumental to nicks and DSB formation and for HR. Fours, unidirectional replication and low efficiency of rARS pose an elevated risk of under- replication.

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27 Numerous studies described factors necessary for the maintenance of the integrity of rDNA array – its absolute size and stability of the repeats number (Saka et al., 2016; Salim et al., 2017). It turns out that up to 10% of the east genes contribute to the maintenance of rDNA integrity albeit to various extents (Saka et al., 2016). Sir2 NAD-dependent histone deacetylase is the main ‘master regulator’ of rDNA integrity and metabolism. Sir2 acts to suppress the recombination at rDNA, inhibits ERC formation and capture, decreases rARS activity, suppresses non-coding transcription in the IGS1 and 2 regions, stimulates cohesin loading (Kobayashi, 2014).

rDNA and yeast aging

The rDNA array stability tightly correlates with the replicative lifespan of yeast cells – the number of times the mother cell could bud off daughter cells before stopping to divide and dying (Kobayashi, 2014). It was initially assumed that the episomal ERCs produced from the rDNA array are causative to shortened lifespan of cells lacking SIR2 (Sinclair and Guarente, 1997), which later was proven to be controversial as other, rDNA non-related, plasmids also showed pro-aging effect (Falcon and Aris, 2003). Notwithstanding, Kobayashi laboratory convincingly showed that inducing rDNA instability by over-activation of the non- coding transcription at rDNA leads to lifespan shortening (Saka et al., 2013).

Conclusion:

Due to repetitive nature, essential role in metabolism, presence of rARS and Fob1-RFB the rDNA array in genetically-amenable budding yeast system is a very attractive model system to study stability of repetitive regions and mechanisms of replication fork progression.

Open questions:

- What is the mechanism of replication fork pausing at Fob1-RFB?

- Whether and how the replication and transcription of rDNA repeats are co- regulated so that the collision between two machineries is minimized?

- How the clustering of active origins in groups of 2-3 at rDNA is maintained?

- What is/are the Sir2 target(s) mediating its broad effect on rDNA metabolism?

- Are ERC just a by-product of rDNA metabolism or have physiological role?

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28 Goals of the thesis

In the course of this thesis I was pursuing the next 3 goals using budding yeast model organism:

(1) to identify the molecular mechanism of Rif1-dependent inhibition of origins firing;

(2) to elucidate Rif1 contribution to maintenance of genome integrity;

(3) to clarify the molecular mechanism of replication fork pausing at proteinaceous barriers.

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29 RESULTS

PART I: Replication initiation is restricted by Rif1-Glc7 and N-terminal region of Mcm4.

Cover page for (Mattarocci et al., 2014)

Prior to our study, Rif1 was recently shown to delay telomere replication in budding yeast (Lian et al., 2011) and regulate replication in fission yeast (Hayano et al., 2012) and mammals (Yamazaki et al., 2012).

In this study (Mattarocci et al., 2014) in parallel with several other laboratories (Dave et al., 2014; Hiraga et al., 2014) we discovered that Rif1 counteracts DDK-dependent origin firing by interacting with PP1 phosphatase (Glc7) and removing activatory phosphorylations from pre-RC components Mcm4 and Sld3.

I joined this study in Shore laboratory after experiments for Figure 2 (Mattarocci et al., 2014) were performed, and contributed to this study by devising and/or performing experiments shown on the figures: Figure 3 (panels A-E), Figure 4 (panel B, left bottom side), Figure S3 (panels A, C-F). Below is our article in its full length.

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Cell Reports

Report

Rif1 Controls DNA Replication Timing in Yeast through the PP1 Phosphatase Glc7

Stefano Mattarocci,1,5Maksym Shyian,1,5Laure Lemmens,1Pascal Damay,1Dogus Murat Altintas,1Tianlai Shi,2,4 Clinton R. Bartholomew,3Nicolas H. Thoma¨,2Christopher F.J. Hardy,3and David Shore1,*

1Department of Molecular Biology and Institute of Genetics and Genomics in Geneva, University of Geneva, 30 quai Ernest-Ansermet, 1211 Geneva, Switzerland

2Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland

3Department of Cell and Developmental Biology, Vanderbilt University Medical Center, T-2212 Medical Center North, Nashville, TN 37232-2175, USA

4Present address: Hoffmann-La Roche Ltd., 4070 Basel, Switzerland

5These authors contributed equally to this work

*Correspondence:david.shore@unige.ch http://dx.doi.org/10.1016/j.celrep.2014.03.010

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARY

The Rif1 protein, originally identified as a telomere- binding factor in yeast, has recently been implicated in DNA replication control from yeast to metazoans.

Here, we show that budding yeast Rif1 protein in- hibits activation of prereplication complexes (pre- RCs). This inhibitory function requires two N-terminal motifs, RVxF and SILK, associated with recruitment of PP1 phosphatase (Glc7). In G1 phase, we show both that Glc7 interacts with Rif1 in an RVxF/SILK- dependent manner and that two proteins impli- cated in pre-RC activation, Mcm4 and Sld3, display increased Dbf4-dependent kinase (DDK) phosphory- lation inrif1mutants. Rif1 also interacts with Dbf4 in yeast two-hybrid assays, further implicating this pro- tein in direct modulation of pre-RC activation through the DDK. Finally, we demonstrate Rif1 RVxF/SILK motif-dependent recruitment of Glc7 to telomeres and earlier replication of these regions in cells where the motifs are mutated. Our data thus link Rif1 to negative regulation of replication origin firing through recruitment of the Glc7 phosphatase.

INTRODUCTION

DNA replication in eukaryotes is initiated from specific chromo- somal sites (origins), which fire in a temporal pattern during S phase that depends on cell type and developmental stage. The unfolding of this replication program is controlled through mech- anisms that remain poorly understood to date. Recent studies show that premature firing of normally late or dormant origins in yeast can lead to activation of a DNA-damage response, most likely as a consequence of deoxynucleotide triphosphate depletion (Mantiero et al., 2011). This finding suggests that con- trol of origin usage may be connected in some way to replication fork progression.

The control of DNA replication initiation is best understood in the budding yeastSaccharomyces cerevisiae, where, unlike in all other eukaryotes studied to date, potential replication origins, or autonomously replicating sequences (ARS), are well defined by a conserved sequence bound constitutively by the origin recognition complex (ORC) (Siddiqui et al., 2013). Origins are first prepared for replication through the loading of the replicative helicase (MCM2–MCM7 hexamer) to form the prereplication complex (pre-RC). Activation of the pre-RC requires the com- bined action of two kinase complexes, the cyclin-dependent kinase (CDK) and the Dbf4-dependent kinase (DDK), the latter consisting of the Cdc7 kinase and its activator Dbf4 (Labib, 2010), and is associated with the recruitment of additional pro- teins, including Cdc45, implicated in MCM2–MCM7 release dur- ing initiation; a set of adaptor proteins, Sld2/Sld3 and Dpb11; the GINS complex, containing four proteins implicated in polymer- ase assembly at the origin; and the leading strand DNA polymer- ase itself, Polε(Araki, 2011).

The temporal pattern of DNA replication during S phase in yeast has been extensively studied (Aparicio, 2013). In this or- ganism, only a small fraction of potential origins actually fire early during S phase. Other origins fire during middle or late S phase or not at all and are thus passively replicated. Significantly, over- expression of several factors, particularly Sld2/Sld3, Dbf4, and Dpb11, accelerates initiation of normally late-firing origins, sug- gesting that the temporal pattern of initiation is entrained by a competition for limiting factors (Mantiero et al., 2011). Telomeres tend to replicate late in S phase (Donaldson, 2005), despite their proximity to nearby ARS elements, due to the action of two pro- teins involved in gene silencing at telomeres, Sir3 and the Ku het- erodimer (Yku70/Yku80) (Cosgrove et al., 2002; Stevenson and Gottschling, 1999). More recent studies have shown that a third telomere-binding protein, Rif1, also determines late telomere replication (Lian et al., 2011). Mutation ofRIF1, in addition to causing earlier telomere replication, also leads to elongation of TG(1–3)tract length at telomeres, yet paradoxically, in other- wise wild-type cells, short telomeric TG-repeat tracts entrain the linked telomere to replicate earlier (Bianchi and Shore, 2007). Taken together, these data implicate the telomere length

62 Cell Reports7, 62–69, April 10, 2014ª2014 The Authors

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