Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate

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Working towards understanding DNA replication

Coupling of a 3’-5’ helicase with a replicative polymerase on a rolling circle substrate

Dissertation

zur Erlangung des akademischen Grades

des Doktors der Naturwissenschaften (Dr. rer. nat.)

an der Universit¨at Konstanz

Mathematisch-naturwissenschaftliche Sektion Fachbereich Biologie

vorgelegt von

Wiebke Galal

Tag der m¨undlichen Pr¨ufung: 15. Mai 2012 Referent: Herr Professor Dr. Mayer Referent: Herr Professor Dr. Scheffner

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-206754

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Acknowlegdements

This project would not have come to life without the mentoring of Dr. Jerard Hurwitz. His guidance, knowledge, language skills – and especially patience – have been invaluable to me in the past years. Thank you! My thanks extend to Dr. Zvi Kelman at the University of Maryland. Zvi and his coworkers have been a constant source of advice and reagents.

I am also grateful for the support I received from everyone in Jerry’s lab. Needless to say that my work relies on the knowledge and support of all lab members. I especially want to thank Dr. Andrea Farina and Dr. Eric Campos for their critical reading of my thesis.

In Konstanz, I would like to thank Dr. Thomas Mayer and Dr. Rolf Knippers for accepting my request for thesis supervision. I am thankful for the help that I received, even though I did not attend any courses at the University of Konstanz. I also want to acknowledge the help, advice and kindness I received from the members of the Mayer lab.

On a more personal note I need to thank my family and friends. My parents Johanna and Joachim and my husband Nader have supported me beyond measure. To list all the support I received from my siblings – Gunnar, Steffen and Antje – would by far exceed this page and my skills in any language. Most of all, I thank Adam for his smile.

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List of Figures

1.1 Model of the prokaryotic replisome . . . 2

1.2 Schematic representation of a rolling circle reaction . . . 5

1.3 Model of the archaeal replisome . . . 6

1.4 Model of the human replisome . . . 9

2.1 GAN interacts with GINS15 . . . 20

2.2 GAN is a Mn²⁺-dependent exonuclease . . . 26

2.3 GAN nuclease produces mononucleotides . . . 27

2.4 GAN is a 5’ → 3’ exonuclease . . . 28

2.5 GAN is an ssDNA-specific exonuclease . . . 29

2.6 GAN nuclease activity is stimulated by GINS15 . . . 30

2.7 Proposed role for GAN during archaeal DNA replication . . . 30

2.8 Plasmid pZLE034 used to transform T. kodakaraensis . . . 31

2.9 Alignment of the amino acid sequence of T. kodakarensis and E. coli . . . 31

2.10 Monomers and dimers of GAN possess nuclease activity . . . 32

2.11 GINS15 stimulates GAN activity . . . 33

2.12 GAN (D34A) interacts with GINS15 . . . 34

2.13 GAN does not degrade RNA in the presence of GINS . . . 35

2.14 GAN is conserved in euryarchaeota . . . 36

3.1 Influence of temperature on Tk primase-catalyzed synthesis of oligonucleotide 43 3.2 Analysis of DNA and RNA products formed by the Tk primase complex . 45 3.3 Template specificity of Tk primase complex . . . 46

3.4 Status of the 5’-termini of RNA and DNA products formed by the Tk DNA primase complex . . . 48

3.5 Effects of dATP and ATP on RNA and DNA synthesis . . . 50

3.6 Primase-dependent replication of M13 DNA . . . 52

3.7 Rolling circle synthesis of leading and lagging strand . . . 53 ix

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3.8 Coupling of Tk DNA Primase activity with Klenow Pol. . . 57

3.9 Oligo dA chains contain 5-phosphate ends . . . 58

3.10 Sequence of oligonucleotides used to construct the 200-nt rolling circle substrate. . . 59

3.11 RNA and DNA synthesis with oligo dT30 as template . . . 59

3.12 Influence of CIP on oligo rA products . . . 59

3.13 TLC separation of products formed after alkaline hydrolyse of ³²P-oligo rA 60 4.1 The hCMG complex is associated with chromatin . . . 64

4.2 Purification of hCMG complex . . . 65

4.3 DNA helicase activity of the hCMG complex on various DNA substrates . 78 4.4 Processivity of the CMG helicase activity . . . 79

4.5 DNA binding activity of the hCMG complex . . . 80

4.6 Rolling circle assay . . . 81

4.7 Isolation of hCMG complex from 293 cells . . . 82

4.8 Western blot analysis of the purified hCMG complex isolated from Sf9 cells 82 4.9 ATPase activity of the hCMG complex . . . 83

4.10 Influence of ATP on the helicase activities of the hCMG and Mcm4/6/7 complexes using M13 substrates . . . 83

4.11 Comparison of the helicase activity of the hCMG and hMcm2-7 complexes 84 4.12 Influence of the 3’ tail sequence on hCMG helicase activity . . . 84

4.13 Influence of ATP levels on the hCMG helicase activity and its binding to oligonucleotide substrates . . . 85

4.14 Purification of Mcm2-7 and Mcm4/6/7 complex . . . 85

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List of Tables

2.1 Proteins that purified with GAN . . . 19 2.2 Oligonucleotides used in this study . . . 25 4.1 Oligonucleotides used in this study . . . 72

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List of abbreviations

abbr. full name bp base pairs

bp/s base pairs per second CIP calf intestine phosphatase CMG Cdc45, Mcm2-7, GINS

C chromatin

CDK cyclin-dependent kinase DDK Dbf4-dependent kinase DUE DNA unwinding element ds double stranded

d drosophila

Dm Drosophila melanogaster

EMSA electrophoretic mobility shift assay Fen1 Flap endonuclease 1

GAN GINS associated nuclease GINS go ichi nii sans

His Histidine

h human

Pi inorganic phosphate PPase inorganic pyrophosphatase PPi inorganic pyrophosphate kb kilobases

mt Methanothermobacter thermautotrophicus Mcm minichromosome maintenance

m mutant

nt nucleotide

nt/s nucleotides per second ORC origin recognition complex Pol polymerase

pre-RC pre-repliative complex

PCNA proliferating cell nuclear antigen

p protein

Pf Pyrococcus furiosus

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abbr. full name

PCNA proliferating cell nuclear antigen

p protein

Pf Pyrococcus furiosus RFC replication factor C RPA replication protein A

RPC replisome progression complex SV40 simian virus 40

ss single stranded

SSB single-strand binding protein Ss Sulfolobus solfataricus SN supernatant

Sld synthetically lethal with Dbp11 Tk Thermococcus kodakaraensis

T tumor

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Summary

DNA replication is an essential process whereby an entire double-stranded DNA is copied to produce a second, identical DNA double helix. This process requires the concerted action of a large number of proteins. The basic mechanism governing this vital macromolecular event is conserved among prokaryotes, archaea and eukaryotes. The helicase unwinds the DNA double helix into two individual strands. Single strand binding proteins coat the single-stranded DNA to prevent the strands from reannealing. Primase is a polymerase that synthesizes the short primers needed to start the replication process.

Primases are necessary because DNA polymerases can only extend a nucleotide chain, not start one. The DNA polymerase starts at the 3’ end of the primer, and, using the original strand as a template, begins to synthesize a new complementary DNA strand by linking the 5’ phosphate group of an incoming nucleotide to the 3’ hydroxyl group at the end of the growing nucleic acid chain.

The studies presented in this thesis are described in three recently published papers.

In the publication titled ”Properties of the human Cdc45/Mcm2-7/GINS helicase complex and its action with DNA polymerase! in rolling circle DNA synthesis”, we describe the purification and biochemical characterization of the human replicative DNA helicase, the CMG complex, which consists of Cdc45, Mcm2-7 and GINS. Another manuscript (”Characterization of the DNA primase complex isolated from the archaea,Thermococcus kodakaraensis”) focuses on the archaeal DNA primase, which consists of a small catalyti- cally active subunit and large regulatory subunit devoid of enzymatic activity. While this structure is similar to the eukaryotic DNA primase, we found that the archaeal complex initiates DNA as well as RNA chainsde novo, while the eukaryotic complex only initiates RNA chains. Finally, we studied the biochemical properties of a previously uncharac- terized nuclease that localizes to the replication components (”A novel DNA nuclease is stimulated by association with the GINS complex”).

Classically, the enzymes involved in replication have been characterized under conditions in which each protein is examined in the absence of other replication components. Rolling circle assays have been recently developed for prokaryotic systems to study the jointly action of replication proteins during DNA synthesis. The primase and the CMG studies presented in this thesis describe the development of an in vitro rolling circle method used to examine leading and lagging strand synthesis with both archaeal and eukaryotic proteins. Our detailed studies in the eukaryotic system revealed that the putative leading strand polymerase!, in concerted action with the helicase, supported the synthesis of long

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DNA chains while DNA polymeraseδ, the putative polymerase involved in lagging strand synthesis, did not support the synthesis of long DNA chains.

Overall, the availability of rolling circle assays will be of great advantage in analyzing the functions played by the various components of the DNA replication machinery of human cells.

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Zusammenfassung

DNA-Replikation ist ein zentraler Prozess, mit dem die gesamte doppelstr¨angige DNA- Helix zu einer zweiten, identischen DNA-Doppelhelix kopiert wird. Bei diesem Vorgang, der bei Bakterien ¨uber Archaeen bis hin zu Eukaryoten beibehalten ist, muss eine Vielzahl von Proteinen zusammenspielen.

Bei der DNA-Replikation wird die DNA-Doppelhelix von der Helikase in zwei unab- hängige Stränge entwunden, die dann durch Einzelstrangbindeproteine bedeckt werden, um das Wiederverschmelzen der Einzelstränge zu verhindern. Die Primase, eine Poly- merase, die Nukleotidketten beginnen kann (während DNA-Polymerasen diese lediglich verlängern können), synthetisiert die für den Replikationsstart notwendigen Primer. Die DNA-Polymerase beginnt dann am 3’-Ende des Primers mit der Synthese eines neuen komplementären DNA-Stranges, bei dem der Originalstrang als Matrize verwendet wird.

Dazu verbinden die Polymerasen die 5’-Phosphatgruppe des neuen Nukleotids mit der 3’- Hydroxylgruppe am Ende der wachsenden Nukleotidkette. Durch die Polarit¨at der DNA kann ein DNA-Strang (der Leitstrang) kontinuierlich, der andere Strang (der Folgestrang) diskontinuierlich repliziert werden.

Die in der vorliegenden Dissertation präsentierten Studien wurden in drei jüngst pub- lizierten Veröffentlichungen beschrieben. Thema der Publikation ”Properties of the human Cdc45/Mcm2-7/GINS helicase complex and its action with DNA polymerase ! in rolling circle DNA synthesis.” ist die Aufreinigung und biochemische Charakterisierung der humanen Replikationshelikase, die aus dem Cdc45-Protein, dem Mcm2-7- und dem GINS- Komplex besteht und kurz CMG-Komplex genannt wird. In einem weiteren Manuskript (”Characterization of the DNA primase complex isolated from the archaeon,Thermococ- cus kodakaraensis.”) wird die archaeelle DNA-Primase behandelt, die aus einer kleinen, katalytisch aktiven und einer großen, regulatorischen Untereinheit besteht. Obwohl die Struktur der archaeellen Primase der der eukaryotischen ähnelt, wurde aufgewiesen, dass die archaeelle Primase Nukleotidketten sowohl mit DNA als auch RNA beginnen kann, während die eukaryotische Primase ausschließlich RNA zum Start von Nukleotidketten verwendet. In ”A novel DNA nuclease is stimulated by association with the GINS complex”

wurden ferner die biochemischen Eigenschaften einer bislang unbekannten, mit Replika- tionsproteinen interagierenden Nuklease untersucht.

Ublicherweise wurden die Enzyme der DNA-Replikation unabhängig voneinander, also in¨ Abwesenheit anderer Replikationsproteine, untersucht. In jüngerer Vergangenheit wurden für eine Reihe prokaryotischer Systeme jedochRolling-Circle-Experimente entwickelt, um

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das Zusammenspiel der verschiedenen replikativen Enzyme auf einem zirkulären DNA- Substrat zu untersuchen. In den in der vorliegenden Arbeit präsentierten Primase- und CMG-Studien wird die Entwicklung einerRolling-Circle-Methode beschrieben, mit der die Leit- und die Folgestrangsynthese durch archaeelle und eukaryotische Proteine untersucht werden können. Durch diese Studie mit dem eukaryotischen System wurde aufgewiesen, dass nur die vermutliche Leitstrangpolymerase !, zusammen mit der humanen Helikase CMG, die Rolling-Circle-Synthese unterstützte, während die, vermutlich den Folgestrang kopierende, DNA-Polymerase δ keine langen DNA-Fragmente im Rolling-Circle-Ansatz produzieren konnte.

Da die Rolling-Circle-Methode nun für die archaeellen und eukaryotischen Systeme zur Verfügung steht, können weitere Zusammenhänge zwischen Replikationsproteinen detail- liert in vitro analysiert werden.

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Chapter 1 Introduction

Successful cell division is dependent on the faithful duplication of its DNA. The basic mechanism of DNA replication is evolutionally conserved. The machinery involved in replication, called the replisome, is initially assembled at specific sites in the genome, called origins that serve as platforms for the recruitment of proteins that form the replisome. Within the replisome, a helicase unwinds the duplex DNA into two strands, des- tined to become leading and lagging strand templates. The DNA polymerases within the replisome duplicate the single-stranded DNA (ssDNA) templates generated by the helicase in a polar manner, as DNA polymerases can only extend DNA chains in the 5’→ 3’ direction. Because of the opposite polarity of leading and lagging strand templates, leading strand synthesis occurs continuously in the 5’→3’ direction while lagging strand synthesis occurs discontinuously in the 3’→5’ direction, thereby producing short Okazaki fragments. The Okazaki fragments are processed and ligated downstream of the replication fork. While leading strand polymerization normally needs to be initiated once at the onset of replication, the discontinuous lagging strand synthesis depends constantly on an initiating polymerase, DNA primase, to start Okazaki fragments. This framework of replication proteins is supported by a plethora of factors that ensure the stability of the replication forks, which are regulated by the cell cycle and influenced by DNA damage.

As many of the proteins involved in DNA replication are essential, their in vivo role in DNA replication is difficult to examine. Thus, sensitive in vitro methods have been developed to study the role of many of the replication factors. The rolling-circle method, first described for prokaryotic systems (reviewed in [1]), is employed here to study components of the archaeal and human replication machineries. The following chapters outline some details of the three domains of life relevant to this work. A wealth of knowledge has been gained for the prokaryotic replication machinery, partly due to the fact that this system has been fully reconstituted in vitro. The archaeal and eukaryotic systems are much less understood because these systems have not as yet been reconstituted in vitro.

The mechanism governing DNA replication in all three systems, however, appears to be highly conserved.

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1.1 Prokaryotic DNA replication

Studies on bacterial systems have benefited from their rapid replication and the isolation of large quantities of replication factors. Due to these advantages a wealth of information on the mechanism of action of their replication factors have accumulated and several groups have been able to reconstitute the bacterial replisome [2, 3]. The prokaryotic core replisome components include the DNA helicase DnaB, the multi-subunit polymerase Pol III holoenzyme, the DNA primase DnaG, and the single-stranded DNA binding protein SSB. Interactions between these factors coordinate their location at the replication fork and stimulate the enzymatic activities (reviewed in [1] and see fig. 1.1).

DnaB DnaG

RNA-Primer

Pol III- holoenzyme

-complex

!"

Fig. 1.1: Model of the prokaryotic replisome. A helicase, DnaB, unwinds the duplex DNA. Pol III holoenzyme duplicates leading and lagging strand starting from primers layed by the primase DnaG. The γ-complex loads the processivity factor β (blue ring) and connects helicase and polymerases via itsτ subunit. Adapted from [4].

Prokaryotic DNA replication starts at a single origin and proceeds bidirectionally around the circular chromosome [5, 6]. TheE. coli origin, oriC, contains several 9-mer sequences, called DnaA boxes, and an adjacent AT-rich region, the DNA unwinding element (DUE) [7, 8]. DnaA, the origin recognition protein binds to the DnaA boxes, leading to the melting of the DUE [9]. Formation of the DnaA-DNA complex supports the loading of the replicative helicase, DnaB [10–12], a ring-shaped hexameric motor protein. The ring encircles one of the two single-stranded DNAs and utilizes the energy derived from ATP hydrolysis to move in the 5’→3’ direction [13, 14]. Upon encountering double-stranded DNA (dsDNA), the helicase continues its movement, thereby separating the two daughter strands [15]. The recruitment of DnaB to origins requires the helicase loading protein,

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1.1. Prokaryotic DNA replication 3 DnaC [16–18]. The single strand DNA binding and the ATPase activities of DnaC are required for this step [16, 19]. DnaC inhibits the helicase activity of DnaB in the ATP- bound state [18, 20] but not in its ADP-bound state [16, 21]. After the dissociation of DnaC, the helicase unwinds DNA and interacts with the DNA primase, DnaG, which leads to the formation of primers [3, 22]. In vivo, DnaB, by virtue of its 5’→3’ polarity, localizes to the lagging strand (see fig. 1.1) and under ideal conditions moves about 1000 base pairs per second (bp/s) when tied to the replisome [2, 23–28]. Its rate of unwinding is significantly slower when assayed in vitro (35bp/s [27]). DnaB accelerates to about 400bp/s when linked to the replicative polymerase [27], and is slowed to 1 to 10 bp/s when coupled to translesion polymerases [29].

The replicative polymerase, Pol III holoenzyme, is composed of 10 different proteins which organize in three subunits, the polymerase core, the processivity factor β and the processivity factor loader γ complex (see fig. 1.1). The number of polymerase cores per holoenzyme is determined by the number of τ proteins per γ complex (see below). The Pol III core is a trimer composed of the α polymerase, the ! and θ subunits [30, 31].

By itself, the α polymerase subunit is inefficient and supports dNTP incorporation at a rate of about 8 nucleotides per second (nts/s). The core is non-processive, as it extends 1-10 nts per binding event [32]. The! subunit is the 3’→5’ exonuclease proofreading unit of Pol III. In the absence of !, the processivity of Pol III holoenzyme is reduced from approximately 50 kb to about 1.5 kb [33], ensuring that the proofreading function trav- els with the replisome. The θ subunit of the core stimulates ! slighty [34]. Its function however, is otherwise unclear to date.

The processivity factor, termed the ’β clamp’, directly binds to the core [35–37]. The clamp enhances the processivity of the polymerase to over 50 kb and increases replication rates to approximately 750nts/s [37]. The β clamp engages the polymerase on primed DNA template through the clamp loader, the γ complex, in an ATP-dependent manner [37–41]. The γ complex is a heptamer of five subunits: γ3δδ^!χψ [42, 43]. The ATPase activity of the complex lies within the γ protein. The interaction to the clamp occurs through theδsubunit of theγ complex [40]. An isoform ofγ, theτ subunit, can substitute the γ protein in the complex. The τ protein is not required for clamp loading but is essential for viability of the organism [44]. It mediates the interaction of the clamp loader with the Pol III core [24, 25]. Thus, the number ofτ subunits perγcomplex determines the number of Pol III cores associated with theγcomplex, with a maximal association of three polymerases [43]. As the replisome requires two polymerases, one on each strand, there are presumably two τ subunits in the γ complex [43, 45, 46]. The single-copy subunits of the γ complex create an intrinsic asymmetry in the complex, which defines a different environment for the associated polymerases in the replisome [47]. These environmental differences are thought to affect the individual polymerases and govern their distinct roles on the leading and lagging strands [3, 46–51].

Lagging strand synthesis requires primers to start Okazaki fragment synthesis. The DNA primase, DnaG, synthesizes short RNA primers that are 10 to 12 nts long [52, 53]. Primase action requires the presence of DnaB [22, 54]. Interactions between SSB adjacent to the primers appears to hold DNA primase at the primers [55–57]. Theχsubunit of the clamp loader competes with primase for the interaction with SSB and thereby accelerates the

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release of primase from the complex [58]. The clamp loader then recruits the β clamp to the primed DNA and thereby attracts the lagging strand polymerase, Pol III holoenzyme, that completes the Okazaki fragment and then detaches from the fragment leaving only a nick between the two adjacent Okazaki fragments [59–62].

This detailed information about replication fork progression was obtained using the rolling- circle method (see fig. 1.2) [3, 52, 63–65]. The rolling-circle assay permits the detailed characterization of joint helicase and polymerase action. Richardson’s group first used this method to describe the viral T7 replication proteins [66]. The rolling-circle DNA substrate provides an annealed primer on a circle. The primer has a 5’ single- stranded DNA overhang and an available 3’-hydroxyl end. The helicase unwinds the duplex DNA in a 5’→3’ direction starting from the flap (see fig. 1.2, left side) which mimics a replication fork. The displacement of the DNA by the helicase generates the single- stranded DNA template for the polymerase to duplicate as it extends the 3’ hydroxyl end of the initial primer. Since the substrate is a circular structure, theoretically there is no end to the reaction, thus the term ’rolling circle’. The 5’ overhang, which gets longer as the reaction proceeds, also acts as a template for the DNA primase and the lagging strand polymerase (see fig. 1.2, right side). Helicase and polymerase are loaded onto primed circular DNA, together with accessory proteins. The advantage of the rolling circle system is that the analysis addresses the concerted action of both helicase and polymerase. Furthermore, the circle contains only three deoxynucleotides. Thus, incorporation of the fourth nucleotide permits the distinction between leading and lagging strand synthesis (see fig. 1.2). All other in vitro methods – with the exception of the SV40 system described in sec. 1.3 – focus on either polymerase or helicase activities, but not both, and thus do not mimic the physiologic condition. Alterations of the template, including structural changes that block replication or nicks, permit the examination of conditions that stall, block or col- lapse replication forks [67, 68]. Since products formed in the rolling circle reaction are very large (over 10 kilobases), the tethering of the rolling- circle substrates to a surface and use of fluorescent nucleotides allows single-molecule analyses [69].

1.2 Archaeal DNA replication

The archaeal domain of life is as diverse in its population as are prokaryotic and eukaryotic. Archaea are classified into two kingdoms, euryarchaea and crenarchaea, based on differences in their 16S rRNA [69]. Relevant for this study is the archaeon Thermococcus kodakaraensis (Tk), which belongs to the kingdom of euryarchaea.

The machinery responsible for archaeal DNA replication appears to have fewer components than their eukaryotic counterparts, but the archaeal and eukaryotic proteins strik- ingly resemble one another in both sequence and structure (reviewed in [71]). The reduced complexity makes the archaeal system attractive for experimental studies.

The mode of archaeal DNA replication, however, resembles that used by prokaryotes.

The genome is organized in a circular chromosome and DNA replication emanates from one origin bi-directionally around the circular template [72, 73]. However, some archaea

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1.2. Archaeal DNA replication 5

5' 5'

Pol III*

clamp clamp

loader

DnaB

dCTP, dGTP

Primase SSB dATP/dTTP [ P]-dNTP

Primer [ P]-dTTP!"#³²

!"#

[ P]-dATP!"#³²

32

Fig. 1.2: Schematic representation of a rolling circle reaction. DnaB unwinds the primer that is annealed to an 100-nucleotide small minicircle and thereby generates single- stranded template for replication. The polymerase extends the primer that the helicase unwinds, and thereby provides double-stranded template for helicase unwinding. Addition of primase initiates lagging strand synthesis on the single-stranded tail that emerges in the leading strand polymerization. The minicircle includes only the nucleotides dCMP, dGMP and dAMP. Leading strand polymerization can therefore be monitored by the incorporation of dTTP and lagging strand polymerization by the incorporation of dATP. Adapted from [70].

species possess two or more putative origins [74–76]. It is tempting to speculate that these origins are used at different times during DNA replication, resembling the eukaryotic usage of early and late origins. Though origins of several archaea have been identified, the origin of the archaea T. kodakaraensis still has yet to be identified.

Little is known about the initiation of replication in archaea. Most sequenced archaeal genomes possess at least one origin binding protein variant, Orc/Cdc6 (reviewed in [77]), named because of its similarity to the eukaryotic proteins Orc and Cdc6. In Pyrococ- cus abyssi the Orc/Cdc6 protein is stably expressed throughout the cell cycle and is associated with chromatin [72]. The replicative helicase, minichromosome maintenance protein (MCM), localizes to the Orc/Cdc6-primed origins at the onset of DNA replication [72]; MCM interacts with Cdc6 [78]. On DNA, both hexamers and double-hexamers have been observed by electron microscopy [79]. In solution, the MCM complex from the euryarchaea Methanothermobacter thermautotrophicus (Mt) was shown to form double hexamers, while the MCM from the crenarchaeal Sulfolobus solfataricus (Ss) exists as a single hexamer [71, 80]. The MCM complexes from the two organisms also differ in their ATPase activity. While the ATPase activity of Mt MCM is stimulated by ss DNA, the activity of Ss MCM is unaffected by DNA [80]. The zinc-binding motifs in the euryarchaeal MCM may explain the difference of properties. Euryarchaeal MCMs possess a zinc-finger motif of the C4 type, that the crenarchaeal MCMs lack [81]. The crenarchaeal MCMs, however, provide a C3 type of putative zinc finger domain [80]. The efficient binding of ssDNA requires the C4 type of zinc-finger domain [82]. The double- and single-hexameric forms of mtMCM both show helicase activity [83]. In contrast to the bacterial helicase DnaB, but like the eukaryotic MCM complex, the archaeal MCM complex has 3’→5’

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helicase directionality, thus it moves along the leading strand template (see fig. 1.3) [82].

The crystal structure of the double-hexameric Mt MCM shows a central cavity in the complex that can encircle double-stranded DNA. Mutations in the central cavity abolish DNA binding [83]. MCM proteins are members of the AAA+ family. Proteins of the AAA+ are remodelers of macromolecular structures whose ATPase domains require amino acid side chains of two adjacent complex subunits. Biochemical studies have established that Mt MCM contains DNA-dependent ATPase activity [84], translocates along ss and ds DNA (reviewed in [85]) and displaces DNA bound proteins [86]. In vivo, the different archaeal species have various copy numbers of MCM variants (reviewed in [77]). Thermococcus kodakaraensis has three different MCM proteins [87]. Two of the MCMs, Tk MCM2 and Tk MCM3, form hexameric structures and display in vitro helicase activity. While the genes for Tk MCM1 and Tk MCM2 could be deleted from the organism, attempts to delete the Tk MCM3 gene were unsuccessful, suggesting an essential character of Tk MCM3. Interactions of MCM to other replicative proteins have been shown. The MCM proteins interact with the GINS complex (Go, ichi, nii, san; Japanese for five, one, two, three) bothin vivo and in vitro (see fig. 1.3). The GINS proteins have been identified in a yeast-two-hybrid screen to MCM inS. solfataricus [88]. The GINS complex is a dimer of a dimer, consisting of two copies of GINS23 and GINS15 [89]. While the GINS complex itself has no enzymatic activity, it interacts with the MCM complex and DNA primase [88–93]. Thus, the GINS complex has been proposed to act as a bridge between one of the lagging strand polymerases and the replicative helicase (fig. 1.3). The GINS complex also interacts with a protein that is a homologue of the bacterial RecJ and the human Cdc45, RecJdbh, inS. solfataricus [88].

PCNA

DNA pol

MCM

GINS

Primase

Primer Lig1

FEN1

SSB

Fig. 1.3: Model of the archaeal replisome. The helicase, MCM, unwinds the duplex DNA ahead of the polymerase (DNA Pol) on the leading strand. The polymerase achieves its processivity due to PCNA. PCNA interacts with lagging strand processing factors, FEN1 and Ligase 1. MCM interacts over GINS indirectly with the DNA primase. Adapted from [77].

The study described in the paper ’A novel DNA nuclease is stimulated by association with the GINS complex’ characterized a nuclease, tk1252p, that localizes at the replication fork [91]. This exonuclease digests ss DNA in the 5’→3’ direction and shares homology with the human Cdc45 protein [94]. Tk1252p interacts with the GINS complex, specifically with

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1.2. Archaeal DNA replication 7 the GINS15 subunit and has been named GAN (GINS associated nuclease). Based on the GAN-GINS interaction and the GAN-Cdc45 homology, Makarova and colleagues proposed that a complex of MCM, GINS and RecJdbh/GAN is a homologue of the human CMG complex (consisting of Cdc45, MCM2-7 and GINS complex), the eukaryotic replicative helicase [94].

After an initial unwinding of the DNA at the origin, the single stranded DNA is bound by the single strand binding (SSB) protein to protect the DNA from damage. Crenarchaeotes cover DNA with an E. coli SSB-like protein complex [95, 96]. The crenarchaeal SSB resembles the structure of the bacterial SSB more than the eukaryotic RPA. Euryarchaeotes possess an RPA-like protein complex. The RPA complex of Pyrococcus furiosus is a het- erotrimer containing subunits of 41, 32 and 14 kDa [97]. Overall, the RPA complexes found in euryarchaea are diverse in size and composition. However, all studied euryarchaea possess at least one p41 subunit and many contain a p32 subunit homologue [98–100]. The structural differences between the RPA- and SSB-like complexes are attributed for the differences in properties. The SSB ofSulfolobus solfataricus interacts with Ss MCM [80], while such interactions have not yet been found for euryarchaeal RPA. On the other side, RPA ofMethanothermobacter thermautotrophicus inhibits the Mt PolB DNA synthesis in the absence of processivity factors [101].

Upstream of the helicase at the replication fork, the leading and lagging strand polymerases duplicate the DNA template. The polymerases require a processivity factor, PCNA (proliferating cell nuclear antigen) that stimulates the polymerase processivity of Thermococcus kodakaraensis polymerase B (Tk PolB) from about 0.5 to about 7 kbp.

PCNA acts as a homotrimer. For its loading on DNA, PCNA requires a loading factor, RFC (replication factor C) [102] which is a pentamer of four copies of the small subunit and one copy of the large subunit, RFCS and RFCL, respectively. RFC loads PCNA preferably on primer-template junctions [1] and requires ATP for its action. Thermococ- cus kodakaraensis possesses two distinct PCNA variants, tk0582 and tk0535, that form homotrimers and stimulate the DNA synthesis activity of Tk PolB [102]. PCNA of S.

solfataricus has been shown to interact with different factors at the replication fork, such as ligase 1 and FEN1, which are both involved in Okazaki fragment maturation. Due to its interaction with the polymerases and downstream processing factors, PCNA has been suggested to act as an organizational platform in addition to its role as a processivity factor [103].

Euryarchaeotes, like T. kodakaraensis, possess two polymerases, D and B, while crenar- chaeotes contain only polymerase B [104, 105]. Polymerase D (PolD) is a dimer of the smaller subunit DP1 and the larger subunit DP2 [106] DP2 alone contains limited polymerase activity [107]. DP1 shows sequence homology to the smaller, noncatalytic, subunits of the eukaryotic replicative polymerases, with Polα(p70), Pol δ (Cdc27p) and Pol

!(p55) [108]. It also harbors a 3’→5’ exonuclease activity, which is highest on mismatched DNA template [109]. The excision of mismatches suggests a role in DNA repair or on the lagging strand in DNA replication [110]. Polymerase B is widely diverse across the different archaeal strains (summarized in [111, 112]). PolB of T. kodakaraensis is monomeric and is highly processive when bound to PCNA [102]. A couple of considerations suggest a role for PolB as the leading strand polymerase in DNA replication. In crenarchaea

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PolB is the only polymerase identified to date [104, 105]. Also, the eukaryotic replicative polymerases belong to the B family of polymerases. Finally, PolB contains a potent 3’→5’

exonuclease proofreading activity [113]. However, which protein acts as the lagging strand polymerase remains unclear.

While the leading strand polymerase only requires one initiation event, the lagging strand polymerase requires priming to initiate each Okazaki fragment. DNA primase mediates priming. Archaea possess two DNA primases, a DnaG-like primase and a eukaryote- like primase. The DnaG-type primase has been demonstrated to play a role in RNA degradation and its gene is dispensable for cell growth [91, 114, 115]. The eukaryote- like primase is a heterodimer of a small p41 and larger p46 subunits. The enzymatic activity lies in the p41 subunit, while the p46 subunit regulates the activity and alters the properties of the catalytic subunit [116]. To date, no homologue of the eukaryotic polymeraseαhas been identified in archaea [117]. The absence of polymeraseαsuggests to that the primase may be responsible for Okazaki fragment initiation prior to the switch to the lagging strand polymerase occurs. The enzymatic properties of the different archaeal primases vary widely. In some archaea, priming with rNTPs has been reported [72, 118, 119]. For Pyrococcus furiosus (Pf), however, the primase initiates DNA synthesis with dNTPs. Also, Pf p41 was able to extend the DNA chains up to several kilobases. In complex with p46 the DNA chains were significantly shorter, the in vitro activity could however be stimulated with ATP. Also, the Pf p41/p46 complex was shown to initiate primers with rNTPs [116].

Readers are referred to the ’Characterization of the DNA primase complex isolated from the archaeon,Thermococcus kodakaraensis’ for the purification and characterization of the Tk primase. This section describes the primer initiation with both dNTPs and rNTPs by the primase complex and the catalytic subunit. The primase complex is required for the primer usage by Tk PolB, the putative replicative polymerase. Current in vitro methods available allow the study of polymerases and helicases separately from each other.

However, under physiological conditions helicase and polymerases interact directly or indirectly, and probably influence each other’s properties. Thus, the second study introduces the rolling circle assay to study the link between replicative helicase, polymerase and primase. In this study, the coupled action of Tk MCM2 and Tk PolB produced fragments longer than 10 kilobases. These long fragments, which mimic leading strands, provided the template for lagging strand synthesis, initiated by the p41/p46 primase complex. Thus, in the presence of all three protein fractions, leading and lagging strand synthesis were observed.

1.3 Eukaryotic DNA replication

Eukaryotic DNA replication is initiated in a tightly regulated manner. In the first step the pre-replicative complex (pre-RC) forms by the step-wise assembly of origin-recognition complex (ORC), Cdc6, Cdt1 and MCM2-7 complex onto chromatin [119]. Electron mi- croscopic studies with purified yeast MCM2-7 showed that the loaded complex encircled

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1.3. Eukaryotic DNA replication 9 double-stranded DNA after it was loaded onto origins [120]. In the second step the pre-RC is activated to a replisome; its activation depends on the activities of the S- phase kinases Dbf7-Cdc7 (DDK) and CDK [121]. The loading of Cdc45 and the GINS complex (a heterotetramer of Sld5, Psf3, Psf2 and Psf1) onto MCM2-7 requires multiple protein factors which minimally include Sld2, Sld3, Dbp11, MCM10, Ctf4 and DNA polymerase (pol)!in yeast [122]. The homologues in higher eukaryotes are presumably ReqQL4 (Sld2), Treslin (Sld3) and TopBP1 (Dbp11). Treslin is essential for cell viability and has been identified through its interaction with TopBP1 in Xenopus [123]. In budding yeast, Sld2 and Sld3 are the only factors involved in the loading process that need to be phosphorylated [122, 124].

The complex of Cdc45, GINS and MCM2-7 (CMG) constitutes the replicative helicase (fig. 1.4) that unwinds origins and contributes to the recruitment of replicative polymerases to the replisome. In vivo experiments in Hela cells showed that the interactions between the CMG complex members only occur at the G1/S transition of the cell cycle and required CDK and DDK activities [125]. The formation of CMG also required a number of the factors that were essential for formation of the yeast replisome progression complex (RPC), such as Ctf4 and TopBP1. The omission or selected degradation of any of the CMG components of the yeast replisome progression complex abolished replication fork movement and displaced other members of the RPC [126, 127]. Also, in the Xenopus cell free system the CMG subunits interacted at stalled replication forks, indicating the presence of a stable complex in this organism [128]. Finally, Botchan’s group isolated the CMG complex fromDrosophila melanogaster (dm) embryos and further characterized the dm CMG complex isolated from the Sf9/Baculovirus system [129, 130]. In vitro the dm CMG complex unwinds DNA in a 3’ → 5’ orientation and thus moves on the leading strand.

PCNA

GINS

Primer pol

pol

RPA

Mcm2-7 Cdc45

pol /primase CMG

!"

Ctf4 Mcm10

Fig. 1.4: Model of the human replisome. The complex of Mcm2-7, Cdc45 and GINS, the CMG, unwinds the duplex DNA ahead of leading and lagging strand polymerase, assigned as Pol$and Polδ, respectively. PCNA increases the processivity of the polymerases. Ctf4 and Mcm10 are representatives of additional factors at the replication fork. Adapted from [131].

The third manuscript enclosed in this thesis describes the isolation and characterization of the human (h) CMG complex from the Sf9/Baculovirus system. As shown with the dm CMG, the h CMG complex requires ATP, magnesium and ss DNA for its loading on

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DNA. Forked DNA structures and dT-rich sequences enhance the 3’→5’ helicase activity of the h CMG complex. Single-strand binding protein from E. coli, as well as its human homolog, hRPA, stimulated the helicase processivity.

The MCM2-7 complex provides the motor of the replicative helicase. The laboratory of Anthony Schwacha showed that yeast MCM2-7 complex possessed in vitro helicase activity using high ionic strength conditions [132]. This helicase activity, however, has not yet been reproduced to date for complexes isolated from higher eukaryotes. The association with Cdc45 and GINS, however, activates the helicase activity ofDrosophila melanogaster MCM2-7 [130]. Since Cdc45 and GINS lack enzymatic activities it is assumed that they activate the helicase activity and participate with the other replicative proteins at the fork.

Previous studies in our laboratory showed that the GINS complex weakly interacts with all three replicative polymerases and stimulates pol! and pol αand to a lesser extent pol δ [133]. This indicates a physical link between the replicative helicase and polymerases.

In yeast, pol ! was shown to associate with replication forks during S phase [134]. It is essential in budding and fission yeast [135, 136]. In human cells, in vivo studies showed that pol ! depletion leads to a more dramatic effect than the depletion of polδ [133].

Human pol ! is a heterotetramer consisting of p261/Pol2, p59, p17 and p12 subunits.

The N-terminus of the p261 subunit is solely responsible for DNA polymerase activity.

In in vitro primer extension assays using the single-stranded DNA plasmid M13mp18, pol ! showed a higher processivity of DNA synthesis than pol δ. Pol δ was originally identified from calf thymus [137] and further characterized using the SV40 replication system (see below) [138]. It is a heterotetramer composed of p124, p66, p51 and p12 (reviewed in [139]). In budding yeast, in vivo studies indicated that pol ! duplicates leading strand while pol δ synthesizes lagging strands (fig. 1.4). Point mutations in the endogenous polymerases lead to distinct misincorporations in the replicated DNA and the travel pattern from known origins of replication was traced by genome-wide analysis using the misincorporations as travel markers [140, 141]. The selective action of these polymerases, pol ! and pol δ, in vitro remains to be established.

The in vitro system using the simian virus (SV) 40 origin DNA with the protein SV40 T Antigen helped identify proteins of the eukaryotic replication fork (reviewed in [142–

145]). In the SV40 replication system, T Antigen assembles as a hexamer at SV40 ori⁺ and unwinds the DNA [146–149]. Thus, T Antigen alone replaces the activities due to the combined action of the bacterial DnaA, DnaC and DnaB as well as the human ORC, Cdc6, Cdt1 and MCM2-7 proteins. Human RPA then binds to the single stranded DNA [150–153]. And although E. coli SSB can substitute for hRPA in DNA binding, hRPA is required for the interaction of T Antigen with human polα/primase complex [151, 154–

157]. The recruited primase catalyzes the synthesis of primers that pol α then extends to fragment lengths of about 40 to 50 nucleotides [158–160]. The clamp loader RFC competes with polα/primase at the primer-template junction and displaces pol α. RFC then loads the clamp PCNA in an ATP-dependent manner and thereby recruits pol δ to the DNA [159, 161, 162]. This system allowed the initial characterization of a number of replication proteins. However, it does not involve the human replicative helicase, as T

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1.3. Eukaryotic DNA replication 11 Antigen serves as both the origin binding protein and helicase. Also, in the SV40 system pol δ replicates both the leading and lagging strand. However, in vivo experiments in Xenopus and human cells showed the requirement for pol ! in DNA replication [133].

In vivo experiments indicate that pol ! crosslinked to human DNA, while it was not crosslinked to SV40 DNA [163].

In the third manuscript initial efforts were undertaken to remodel the human replication fork. Using a primed minicircle, we showed that the coupling of the hCMG with human pol!, but not pol δ, formed fragments that are larger than 10 kilobases. The production of these large fragments is in keeping with the action of leading strand polymerization.

The results also suggest that pol ! may directly contact the CMG complex and increase its processivity.

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Chapter 2 A novel DNA nuclease is stimulated by association with the GINS

complex

Zhuo Li,^1,2 Miao Pan,^1,2 Thomas J. Santangelo,³ Wiebke Chemnitz,⁴ Wei Yuan,^1,5 James L. Edwards,^1,5 Jerard Hurwitz,⁴ John N. Reeve,³ and Zvi Kelman^1,2,^∗

Nucleic Acids Res. 2011 August; 39(14): 6114–6123.

1Institute for Bioscience and Biotechnology Research, 9600 Gudelsky Drive, Rockville, MD 20850

2Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742

3Department of Microbiology, Ohio State University, Columbus, OH 43210

4Program in Molecular Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065

5Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742, USA

*To whom correspondence should be addressed.

13

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2.1 Abstract

Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharac- terized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a processive, single-strand DNA-specific exonuclease that degrades DNA in the 5’→3’ direction.

TK1252p binds specifically to the GINS15 subunit ofT. kodakaraensisGINS complex and this interaction stimulates the exonuclease activity in vitro. This novel archaeal nucle- ase, designated GINS-associated nuclease (GAN), also forms a complex in vivo with the euryarchaeal-specific DNA polymerase D. Roles for GAN in replisome assembly and DNA replication are discussed.

2.2 Introduction

Chromosomal DNA replication has many universally conserved features, but there are differences in the proteins and complexes that initiate and maintain DNA replication forks in Bacteria,Archaea and Eukarya. Many of the proteins required for bacterial and eukaryal replication have been isolated and characterized extensively, while most of the components of the archaeal replication machinery have only been putatively identified by bioinformatics. When archaeal proteins with sequences in common with bacterial and/or eukaryal replisome proteins have been investigated, the results obtained have generally confirmed their predicted replication functions [71, 77, 164]. This in silico approach, however, does not readily identify archaeal-specific replisome proteins. To address this limitation,∼20Thermococcus kodakaraensisstrains were constructed, each of which synthesized an established archaeal replication protein with an amino- or carboxy- terminal hexahistidine extension (His6-tag). These proteins were purified directly by nickel-affinity fromT. kodakaraensiscell lysates, and all proteins that were consistently co-isolated with each His6-tagged replication protein were identified [91]. Here, we report the characterization of a novel archaeal nuclease, encoded byTK1252, that was present in the complexes isolated by nickel-binding of His6-tagged subunits of the T. kodakaraensis GINS complex and the archaeal-specific DNA polymerase D (Pol D).

In Eukarya, the heterotetramer GINS complex (containing Sld5, Psf1, Psf2 and Psf3) associates with the mini-chromosome maintenance (MCM) proteins, Mcm2-7 and with Cdc45 to form the Cdc45, Mcm2-7, GINS (CMG) complex. This complex has a 3’→ 5’

DNA helicase activity and is thought to function as the replicative helicase [126, 129, 165].

The GINS complex is required to establish and maintain replication forks [166–168] and also interacts with the Polα-primase complex that synthesizes primers on the lagging strand [89, 165]. With these features, the eukaryal GINS complex appears to be the func- tional homologue of the ¨IˇD subunit (DnaX) of the Escherichia coli replisome that binds to the bacterial replicative DNA polymerase (Pol III), DNA helicase (DnaB) and primase (DnaG) at the replication fork and coordinates leading and lagging strand syntheses

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2.3. Materials and Methods 15 [169]. Sequence homologies predict that manyArchaea, includingT. kodakaraensis, have a GINS complex assembled from two molecules each of GINS15 (TK0536p) and GINS23 (TK1619p), proteins most closely related to the eukaryal Psf1 and Sld5, and Psf2 and Psf3 proteins, respectively [89, 90]. Consistent with GINS being an archaeal replisome component, investigations of [GINS152–GINS232] complexes from several Archaea have documented interactions with the archaeal primase, MCM, Pol D and PCNA [88, 91–93].

In theT. kodakaraensis genome annotation, the protein encoded by TK1252 is predicted to be a single-strand specific nuclease [170]. The results reported here confirm that this protein does associate with the GINS complex, specifically with the GINS15 component, and demonstrate that it is a single-strand (ss) DNA-specific 5’→3’ exonuclease. The exonuclease activity of this protein, designated GINS-associated nuclease (GAN), is stimulated by its interaction with GINS15. Possible roles for the GAN–GINS association during archaeal DNA replication are discussed.

2.3 Materials and Methods

2.3.1 Nuclease substrates

[γ−³²P]ATP was purchased from Perkin Elmer. Unlabeled, Cy3- and Cy5-labeled deoxy- and ribo-oligonucleotides, with the sequences listed in Supplementary (tab. 2.2), were obtained from the NIST/UMD nucleic acids synthesis facility. Double-strand (ds) DNA substrates were generated by annealing complementary oligonucleotides followed by PAGE purification, as previously described [171]. To obtain linear and circular 200-mer substrates, 1.5 nmol of the 100-mer oligonucleotides A and B (Supplementary tab. 2.2) were phosphorylated by incubation with 40 U of T4 polynucleotide kinase for 1 h at 37^◦C.

The phosphorylation reaction mixture for oligonucleotide B also contained 71 pmol of [γ−³²P]ATP. To construct the linear substrate, 0.5 nmol of phosphorylated oligonucleotides A and B plus 2.5 nmol of the bridge oligonucleotide AB (Supplementary tab. 2.2) were mixed in 20 mM HEPES (pH 7.5), 150 mM NaCl, heated to 100^◦C and the mixture was then allowed to cool slowly to 22^◦C. This procedure was also used to generate the circular substrate, except that the reaction mixture also contained 2.5 nmol of the bridge oligonucleotide BA (Supplementary tab. 2.2). The reaction mixtures were placed at 16^◦C, 8000 U of T4 DNA ligase were added and incubation continued for 14 h. The reaction products were separated by electrophoresis at 15 W for 75 min through 10% (w/v) polyacrylamide–

8 M urea gels run in TBE. The regions of the gel containing the desired 200-mer linear and circular ssDNAs were excised and the DNAs eluted from the gel into 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, ethanol precipitated and dissolved in 30µl of TE (pH 8). The resulting solution was passed through a S300 mini-column filter (GE Healthcare).

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2.3.2 Plasmids construction

For protein expression in E. coli, the genes encoding GAN (TK1252), GINS15 (TK0536) and GINS23 (TK1619) were PCR-amplified from T. kodakaraensis genomic DNA using primers (listed in Supplementary tab. 2.2) that added an in-frame His6-encoding sequence to the 3’-terminus of the amplified gene. The amplified DNAs were ligated with pET15b (TK1252) or pET21a (TK0536 and TK1619) linearized by digestion with the restric- tion enzyme listed in Supplementary tab. 2.2. A plasmid that directed the synthesis of GAN (D34A) was generated by site-specific mutagenesis from the plasmid that expressed TK1252 by using a QuikChange mutagenesis kit (Stratagene) using oligonucleotides with the sequences listed in Supplementary tab. 2.2.

To construct a T. kodakaraensis strain that synthesized GAN-His₆ in vivo, TK1252 and DNA from immediately upstream and downstream of TK1252 were separately amplified from T. kodakaraensis genomic DNA. An overlapping PCR was used to add an His6- encoding sequence in-frame to the 5’-terminus of TK1252 [172]. The three amplified DNAs were cloned into pUMT2 [173] adjacent to trpE (TK0254) to generate plasmid pZLE034 (Supplementary fig. 2.9). In pZLE034, TK1252-His6 is positioned between genomic sequences that are homologous to the DNA immediately upstream and downstream of TK1252 in the T. kodakaraensis KW128 genome. An aliquot of pZLE034 DNA was used to transformT. kodakaraensis KW128 (∆pyrF; ∆trpE::pyrF) as previously described [173, 174] and transformants were selected by growth on plates lacking tryptophan. The desired replacement of TK1252 with the GAN-His6 encoding gene was confirmed in a representative transformant, designated T. kodakaraensis 34-5, by diagnostic PCR and sequencing [91].

2.3.3 Recombinant protein purification

The plasmids encoding GAN, GAN (D34A), GINS15 or GINS23 were transformed into E. coli BL21 (DE3)-CodonPlus-RIL (Stratagene). Isopropyl-β-d-thiogalactopyranoside induction, expression at 16^◦C for 16 h and purification of the recombinant N-terminal His6-tagged GAN and GAN (D34A), and C-terminal His6-tagged GINS15 and GINS23 from E. coli cell lysates by Ni²⁺-affinity chromatography were carried out as previously described [175]. Aliquots of the purified proteins were stored at −80^◦C.

2.3.4 Size exclusion chromatography

Aliquots of each experimental protein (100µg) or protein mixture and Gel Filtration standards (Bio-Rad) were dissolved in 200µl of 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10% (v/v) glycerol and loaded onto a Superdex-200 column (HR10/30; GE Healthcare) pre-equilibrated in the same buffer. Fractions (250µl) were collected from the column at a flow rate of 0.5 ml/min. The proteins present in aliquots (80µl) of each fraction were separated by electrophoresis through a 12% (w/v) polyacrylamide-SDS gel and stained with Coomassie brilliant blue (R250).

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2.3. Materials and Methods 17

2.3.5 Nuclease assays

Unless otherwise noted in the figure legends, the nuclease assay reaction mixtures (20µl) containing the DNA substrate, BSA (125µg/ml), 25 mM Tris-HCl (pH 7.5), 2 mM MnCl2

and GAN, were incubated at 70^◦C for 20 min. Nuclease digestion was stopped by adding 20µl of 95% formamide, 0.1× TBE, 10 mM EDTA and incubation at 100^◦C for 2 min.

The digestion products were visualized and quantified by phosphorimaging after electrophoretic separation through 20% (w/v) polyacrylamide-8 M urea gels run in TBE for 1.25 h at 15 W. For native gels nuclease reactions were stopped by adding 5µl of 50% glycerol, 20 mM EDTA. The digestion products were visualized and quantified by phosphorimaging after electrophoretic separation through 20% (w/v) polyacrylamide gels run in TBE for 2 h at 300 V.

2.3.6 Liquid chromatography-mass spectrometry

Aliquots (10µM) of the DNA templates, 5’-AAAAAAGG and 5’-GGAAAAAA, were incubated in reaction mixtures (50µl), with or without 20 pmol GAN, for 1 h at 70^◦C in a buffer containing 5 mM ammonium formate (pH 6.5), 2 mM MnCl2. The products were subjected to liquid chromatography (LC)/mass spectrometry (MS) analyses using the neg- ative ion mode with a Finnigan LTQ ion trap mass spectrometer (San Jose, CA, USA) equipped with nanospray ionization (NSI) interface coupled to an Agilent 1200 HPLC system (Palo Alto, CA, USA). The flow from the Aligent pump was split from 0.85 ml to 25 nl/min using a 75µm internal diameter (ID) silica capillary as the flow splitter.

Separations were performed using 50µm ID silica capillary columns (Polymicro Technol- ogy, Phoenix, AZ, USA) with in-house made frit packed with 15 cm of 3µm Atlantis T3 C18 aqueous reversed phase particles (Waters, Milford, MA, USA). The mobile phase A was 5 mM ammonium formate (pH 6.0) in water, and the mobile phase B was 5 mM ammonium formate in methanol. Analytes were eluted over 30 min using a 0−95% linear gradient of solvent B. The heated capillary was at 200^◦C. Fragmentation was activated by collision-induced dissociation of 35%. Selective reaction monitoring was implemented with the following transitions: dAMP: 330.1 to 195.1 m/z; dGMP: 346.1 to 195.1 m/z;

dAMP-dAMP: 643.1 to 330.1 m/z; and dGMP-dGMP: 675.1 to 346.1 m/z. The instru- ment control, data acquisition, and data analysis were performed by Xcalibur software (Thermo Electron Corporation, version 2.0.7 SP1).

2.3.7 Isolation and identification of His

6

-tagged GAN and asso- ciated proteins from T. kodakaraensis

Thermococcus kodakaraensis 34-5 cultures (5 l) were grown to late exponential phase (OD600 of ∼0.8) at 80^◦C in MA-YT medium supplemented with 5 g sodium pyruvate/l in a BioFlow 415 fermentor (New Brunswick Scientific). The cells were harvested by centrifugation, resuspended in 30 ml of buffer A [25 mM Tris-HCl (pH 8), 500 mM NaCl, 10 mM

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imidazole and 10% glycerol] and lysed by sonication. After centrifugation, the resulting clarified lysate was loaded onto a 1 ml HiTrap chelating column (GE Healthcare) pre- equilibrated with NiSO4. The column was washed with buffer A and proteins were eluted using a linear imidazole gradient from buffer A to 67% buffer B [25 mM Tris-HCl (pH 8), 100 mM NaCl, 150 mM imidazole and 10% glycerol]. Fractions that contained the GAN protein were pooled and dialyzed against buffer C [25 mM Tris-HCl (pH 8), 500 mM NaCl, 0.5 mM EDTA, 2 mM DTT]. Aliquots (30µg) of the protein were precipitated by adding trichloroacetic acid (TCA; 15% final concentration). The TCA- precipitated proteins were identified by multi-dimensional protein identification technology at the Ohio State Uni- versity mass spectrometry facility (http://www.ccic.ohio-state.edu/MS/proteomics.htm) using the MASCOT search engine. The protein isolation and mass spectrometry analyses were also repeated twice using lysates from two independent cultures ofT. kodakaraensis KW128. These control experiments identified the T. kodakaraensis proteins that bound and eluted from a Ni²⁺- charged matrix in the absence of a His6-tagged protein. All proteins co-isolated with His6-GAN by binding to Ni²⁺-matrix fromT. kodakaraensis 34- 5 cell lysates that had high MASCOT scores, and that were not present in the control samples, are listed in tab. 2.1.

Proteins with at least two peptides matches are listed with their molecular weight, MAS- COT score, and the percentage of the amino acid sequence covered by the matching peptides (see text for further details).

2.4 Results

2.4.1 Purified GAN and GINS15 form complexes in solution

GAN (TK1252p) was co-isolated with His₆-GINS15 (TK0536p) fromT. kodakaraensis cell lysates by His6-GINS15 binding to a Ni²⁺-charged matrix followed by imidazole elution consistent with GAN forming a stable complex with GINS15 in vivo [91]. To determine if these proteins also interacted in vitro, recombinant GAN and GINS15 were mixed and the products examined by size exclusion chromatography. In the absence of GINS15, the GAN (52.9 kDa) elution profile was consistent with the presence of monomers and (GAN)2 dimers (fig. 2.1 (A); elution peaks in fractions 53 and 59). GINS15 (21.5 kDa) alone eluted almost exclusively at a position consistent with a (GINS15)2 dimer (fig. 2.1 (B), elution peak in fraction 56), as reported previously for GINS15 fromSulfolobus solfataricus [90].

When incubated together, GAN and GINS15 interacted to form several complexes that eluted in fractions consistent with the formation of complexes larger than (GAN)2 and (GINS15)2 dimers (Figure 1E, elution peaks in fractions 38 and 47). Incubation of GAN with GINS23 (19.2 kDa) did not result in the formation of larger complexes (fig. 2.1 (F)). GAN bound the [GINS152-GINS232] complex (fig. 2.1 (G)), suggesting that the GAN-GINS15 interactions did not disrupt the GINS complex [90, 92].

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2.4. Results 19

Tab. 2.1: Proteins that purified with GAN

Gene # Score MW (Da) Peptide Percent Function matches coverage

TK1903 1354 150 190 54 19.5 Pol D-L

TK1902 947 80 848 43 28.4 Pol D-S

TK1252 746 52 858 26 37.1 GAN

TK1619 155 19 154 6 14.6 GINS23

TK1637 105 29 250 4 13.8 Proteasome subunit alpha

TK1496 97 22 992 3 12.9 30S ribosomal protein S2

TK0536 87 21 583 2 14.9 GINS15

TK1748 81 125 718 3 3.5 Isoleucyl-tRNA synthetase

TK0847 79 8671 4 38.6 Hypothetical protein TK0847

TK2217 79 44 001 2 3.3 2-amino-3-ketobutyrate

coenzyme A ligase

TK0593 73 45 867 3 6.7 Unknown

TK2157 54 36 667 3 6 Unknown

TK2106 50 46 763 2 3.5 Phosphopyruvate hydratase

TK1940 48 39 764 3 11.2 Small-conductance

mechanosensitive channel

TK0171 48 41 290 2 2.8 Unknown

TK0566 45 96 249 2 1.6 DEAD/DEAH box RNA helicase

TK0714 43 74 602 3 2.7 Iron(II) transport protein B

TK2253 41 28 743 13 3.2 Unknown

TK0470 40 198 006 2 0.6 Reverse gyrase

TK2276 39 23 395 2 16.9 Orotidine 5’-phosphate

decarboxylase

TK1448 37 39 518 2 2 5,10-methylenetetrahydrofolate

reductase

TK2270 29 7456 2 30.2 Unknown

TK2255 28 48 783 2 3 Bifunctional phosphatase/dolichol-

phosphate glucosyltransferase

TK0263 27 43 206 2 3.5 3-phosphoshikimate

1-carboxyvinyltransferase

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Fig. 2.1: GAN interacts with GINS15. A sample (100µg) of each protein listed to the right of the corresponding panels (A through G) was subjected to Superdex-200 gel filtration analysis. Aliquots (80µl) from each fraction were separated by electrophoresis through 12%

polyacrylamide-SDS gels and stained with Coomassie brilliant blue (R-250). The fractions in whichγ-globulin (158 kDa), ovalbumin (44 kDa) and myoglobin (17 kDa) eluted are noted at the top of the figure.

2.4.2 GAN is a ssDNA nuclease

Based on limited sequence similarities between GAN and E. coli RecJ (Supplementary fig. 2.9), GAN was predicted to be a ssDNA nuclease [170]. As shown in fig. 2.2 (A), this

Working towards understanding DNA replication : coupling of a 3'-5' helicase with a replicative polymerase on a rolling circle substrate